Polynucleotide encoding coleopteran-toxic polypeptide

ABSTRACT

Disclosed are novel insecticidal polypeptides, and compositions comprising these polypeptides, peptide fragments thereof, and antibodies specific therefor. Also disclosed are vectors, transformed host cells, and transgenic plants that contain nucleic acid segments that encode the disclosed δ-endotoxin polypeptides. Also disclosed are methods of identifying related polypeptides and polynucleotides, methods of making and using transgenic cells comprising these polynucleotide sequences, as well as methods for controlling an insect population, such as Colorado potato beetle, southern corn rootworm and western corn rootworm, and for conferring to a plant resistance to a target insect species.

This application is a divisional of application Ser. No. 10/408,692,filed Apr. 7, 2003 now U.S. Pat. No. 7,078,592, which is a divisional ofapplication Ser. No. 09/563,269, filed May 3, 2000, now U.S. Pat. No.6,555,655, which claims the benefit of U.S. Provisional Application No.60/172,240, filed May 4, 1999.

1.0 BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of molecularbiology. More particularly, certain embodiments concern methods andcompositions comprising DNA segments, and proteins derived frombacterial species. More particularly, it concerns novel genes fromBacillus thuringiensis encoding coleopteran-toxic crystal proteins.Various methods for making and using these DNA segments, DNA segmentsencoding synthetically-modified δ-endotoxin polypeptides, and native andsynthetic crystal proteins are disclosed, such as, for example, the useof DNA segments as diagnostic probes and templates for proteinproduction, and the use of proteins, fusion protein carriers andpeptides in various immunological and diagnostic applications. Alsodisclosed are methods of making and using nucleic acid segments in thedevelopment of transgenic plant cells containing the polynucleotidesdisclosed herein.

1.2 Description of the Related Art

Because crops of commercial interest are often the target of insectattack, environmentally-sensitive methods for controlling or eradicatinginsect infestation are desirable in many instances. This is particularlytrue for farmers, nurserymen, growers, and commercial and residentialareas which seek to control insect populations using eco-friendlycompositions. The most widely used environmentally-sensitiveinsecticidal formulations developed in recent years have been composedof microbial pesticides derived from the bacterium Bacillusthuringiensis. B. thuringiensis is a Gram-positive bacterium thatproduces crystal proteins or inclusion bodies which are specificallytoxic to certain orders and species of insects. Many different strainsof B. thuringiensis have been shown to produce insecticidal crystalproteins. Compositions including B. thuringiensis strains which produceinsecticidal proteins have been commercially-available and used asenvironmentally-acceptable insecticides because they are quite toxic tothe specific target insect, but are harmless to plants and othernon-targeted organisms.

1.2.1 δ-Endotoxins

δ-endotoxins are used to control a wide range of leaf-eatingcaterpillars and beetles, as well as mosquitoes. These proteinaceousparasporal crystals, also referred to as insecticidal crystal proteins,crystal proteins, Bt inclusions, crystalline inclusions, inclusionbodies, and Bt toxins, are a large collection of insecticidal proteinsproduced by B. thuringiensis that are toxic upon ingestion by asusceptible insect host. Over the past decade research on the structureand function of B. thuringiensis toxins has covered all of the majortoxin categories, and while these toxins differ in specific structureand function, general similarities in the structure and function areassumed. Based on the accumulated knowledge of B. thuringiensis toxins,a generalized mode of action for B. thuringiensis toxins has beencreated and includes: ingestion by the insect, solubilization in theinsect midgut (a combination stomach and small intestine), resistance todigestive enzymes sometimes with partial digestion actually “activating”the toxin, binding to the midgut cells, formation of a pore in theinsect cells and the disruption of cellular homeostasis (English andSlatin, 1992).

One of the unique features of B. thuringiensis is its production ofcrystal proteins during sporulation which are specifically toxic tocertain orders and species of insects. Many different strains of B.thuringiensis have been shown to produce insecticidal crystal proteins.Compositions including B. thuringiensis strains which produce proteinshaving insecticidal activity against lepidopteran and dipteran insectshave been commercially available and used as environmentally-acceptableinsecticides because they are quite toxic to the specific target insect,but are harmless to plants and other non-targeted organisms.

The mechanism of insecticidal activity of the B. thuringiensis crystalproteins has been studied extensively in the past decade. It has beenshown that the crystal proteins are toxic to the insect only afteringestion of the protein by the insect. The alkaline pH and proteolyticenzymes in the insect mid-gut solubilize the proteins, thereby allowingthe release of components which are toxic to the insect. These toxiccomponents disrupt the mid-gut cells, cause the insect to cease feeding,and, eventually, bring about insect death. For this reason, B.thuringiensis has proven to be an effective and environmentally safeinsecticide in dealing with various insect pests.

As noted by Höfte et al., (1989) the majority of insecticidal B.thuringiensis strains are active against insects of the orderLepidoptera, i.e. caterpillar insects. Other B. thuringiensis strainsare insecticidally active against insects of the order Diptera, i.e.,flies and mosquitoes, or against both lepidopteran and dipteran insects.In recent years, a few B. thuringiensis strains have been reported asproducing crystal proteins that are toxic to insects of the orderColeoptera, i.e., beetles (Krieg et al., 1983; Sick et al., 1990;Donovan et al., 1992; Lambert et al., 1992a; 1992b).

1.2.2 Genes Encoding Crystal Proteins

Many of the δ-endotoxins are related to various degrees by similaritiesin their amino acid sequences. Historically, the proteins and the geneswhich encode them were classified based largely upon their spectrum ofinsecticidal activity. The review by Höfte and Whiteley (1989) discussesthe genes and proteins that were identified in B. thuringiensis prior to1990, and sets forth the nomenclature and classification scheme whichhas traditionally been applied to B. thuringiensis genes and proteins.cryI genes encode lepidopteran-toxic CryI proteins, and cryII genesencode CryII proteins that are toxic to both lepidopterans anddipterans. cryIII genes encode coleopteran-toxic CryIII proteins, whilecryIV genes encode dipteran-toxic CryIV proteins.

Based on the degree of sequence similarity, the proteins were furtherclassified into subfamilies; more highly related proteins within eachfamily were assigned divisional letters such as CryIA, CryIB, CryIC,etc. Even more closely related proteins within each division were givennames such as CryIC1, CryIC2, etc.

Recently, a new nomenclature was developed which systematicallyclassified the Cry proteins based upon amino acid sequence homologyrather than upon insect target specificities (Crickmore et al., 1998).The classification scheme for many known toxins, not including allelicvariations in individual proteins, is summarized in Section 4.3.

1.2.3 Crystal Proteins Toxic to Coleopteran Insects

The cloning and expression of the cry3Bb gene has been described(Donovan et al., 1992). This gene encodes a 74-kDa protein havinginsecticidal activity against Coleopterans, such as Colorado potatobeetle (CPB), and southern corn root worm (SCRW).

A B. thuringiensis strain, PS201T6, reported to have activity againstwestern corn rootworm (WCRW, Diabrotica virgifera virgifera) wasdescribed in U.S. Pat. No. 5,436,002 (specifically incorporated hereinby reference in its entirety). This strain also showed activity againstMusca domestica, Aedes aegypti, and Liriomyza trifoli.

The cloning and expression of the cryET29 gene has also been described(Intl. Pat. Appl. Publ. Ser. No. WO 97/17507, 1997). This gene encodes a25-kDa protein that is active against Coleopteran insects, particularlythe CPB, SCRW, WCRW, and the cat flea, Ctenocephalides felis.

The cloning and expression of the cryET33 and cryET34 genes has beendescribed (Intl. Pat. Appl. Publ. Ser. No. WO 97/17600, 1997). Thesegenes encode proteins of ˜30 and ˜15 kDa, respectively, and are activeagainst Coleopteran insects, particularly CPB larvae and the Japanesebeetle (Popillia japonica).

The vip1A gene, which produces a vegetative, soluble, insecticidalprotein, has also been cloned and sequenced (Intl. Pat. Appl. Publ. Ser.No. WO 96/10083, 1996). This gene encodes a protein of approximately 80kDa, that is active against both WCRW and northern corn rootworm (NCRW).

Another endotoxin active against coleopteran insects, including WCRW, isCry1Ia (Intl. Pat. Appl. Publ. Ser. No. WO 90/13651, 1990). The geneencoding this 81-kDa polypeptide has been cloned and sequenced.

Additional crystal proteins with toxicity towards the WCRW have beendescribed (Intl. Pat. Appl. Publ. Ser. No. WO 97/40162, 1997). Theseproteins appear to function as binary toxins and show sequencesimilarity to mosquitocidal proteins isolated from B. sphaericus.

Certain strains of B. sphaericus are highly active against mosquitolarvae, with many producing, upon sporulation, a crystalline inclusioncomposed of two protein toxins. The analysis of the genes encoding theseproteins have been described by Baumann et al., (1988). The toxins aredesignated P51 and P42 on the basis of their predicted molecular massesof 51.4- and 41.9-kDa, respectively. The P42 protein alone is weaklyactive against mosquito larvae. The P51 protein has no mosquitocidalactivity by itself. Both P51 and P42 are required for full insecticidalactivity. There are no reports of the crystal proteins of B. sphaericushaving activity on any insects other than mosquitos (for a recent reviewsee Charles et al., 1996a; 1996b).

A second class of mosquitocidal protein toxins are produced by somestrains of B. sphaericus. These proteins, known as Mtx toxins, areproduced during vegetative growth and do not form a crystallineinclusion. The two Mtx toxins that have been identified, designated Mtxand Mtx2, have molecular masses of 100 and 30.8 kDa, respectively. Thecloning and sequencing of the genes for these toxins, designated mtx andmtx2, has been described (Thanabalu et al., 1991, Thanabalu and Porter,1995). The Mtx and Mtx2 proteins do not share sequence similarity to anyother known insecticidal proteins, including the crystal proteins of B.sphaericus and B. thuringiensis.

2.0 SUMMARY OF THE INVENTION

The present invention provides novel insecticidal polypeptides and DNAsequences that encode them. For five of these polypeptides, theirdisimiliarity to the known crystal proteins indicates the existence of anew class or sub-class of B. thuringiensis crystal proteins, as theyshare less than 65% amino acid sequence identity with any of thepresently known insecticidal polypeptides. The invention furtherprovides novel polypeptides that when in combination, produceinsecticidally-active crystal proteins. Also provided are transformedhost cells, transgenic plants, vectors, and methods for making and usingthe novel polypeptides and polynucleotides.

In a first embodiment, the invention provides an isolated CryET69polypeptide comprising at least 7 contiguous amino acids from SEQ IDNO:14. More preferably the polypeptide comprises at least 9 or at least11 contiguous amino acids from SEQ ID NO:14. Still more preferably, thepolypeptide comprises at least 13 or at least 15 contiguous amino acidsfrom SEQ ID NO:14, and more preferably comprises at least 17 or at least19 contiguous amino acids from SEQ ID NO:14. In an exemplary embodiment,the polypeptide comprises the sequence of SEQ ID NO:14. Such apolypeptide is preferably encoded by a nucleic acid segment thatcomprises an at least 45-basepair contiguous nucleotide sequence fromSEQ ID NO:13, and more preferably is encoded by a nucleic acid segmentthat comprises an at least 90-basepair contiguous sequence from SEQ IDNO:13. More preferably still, such a polypeptide is encoded by a nucleicacid segment that comprises an at least 150-basepair contiguous sequencefrom SEQ ID NO:13. Exemplary polynucleotides encoding the insecticidalpolypeptide comprise an at least 300-basepair contiguous nucleotidesequence from SEQ ID NO:13, and in one embodiment comprises thenucleotide sequence of SEQ ID NO:13.

Also disclosed and claimed is an isolated CryET84 polypeptide comprisingat least 15 contiguous amino acids from SEQ ID NO:19. More preferablythe polypeptide comprises at least 30 to 45 contiguous amino acids fromSEQ ID NO:19. Still more preferably, the polypeptide comprises at least45 to 90 contiguous amino acids from SEQ ID NO:19, and more preferablycomprises at least 90 to 150 contiguous amino acids from SEQ ID NO:19.In an exemplary embodiment, the polypeptide comprises the sequence ofSEQ ID NO:19. Such a polypeptide is preferably encoded by a nucleic acidsegment that comprises an at least 45-basepair contiguous nucleotidesequence from SEQ ID NO:18, and more preferably is encoded by a nucleicacid segment that comprises an at least 90-basepair contiguous sequencefrom SEQ ID NO:18. More preferably still, such a polypeptide is encodedby a nucleic acid segment that comprises an at least 150-basepaircontiguous sequence from SEQ ID NO:18. Exemplary polynucleotidesencoding the insecticidal polypeptide comprise an at least 300-basepaircontiguous nucleotide sequence from SEQ ID NO:18, and in one embodimentcomprises the nucleotide sequence of SEQ ID NO:18.

Also disclosed and claimed is an isolated CryET75 polypeptide comprisingat least 15 contiguous amino acids from SEQ ID NO:16. More preferablythe polypeptide comprises at least 30 to 45 contiguous amino acids fromSEQ ID NO:16. Still more preferably, the polypeptide comprises at least45 to 90 contiguous amino acids from SEQ ID NO:16, and more preferablycomprises at least 90 to 150 contiguous amino acids from SEQ ID NO:16.In an exemplary embodiment, the polypeptide comprises the sequence ofSEQ ID NO:16. Such a polypeptide is preferably encoded by a nucleic acidsegment that comprises an at least 45-basepair contiguous nucleotidesequence from SEQ ID NO:15, and more preferably is encoded by a nucleicacid segment that comprises an at least 90-basepair contiguous sequencefrom SEQ ID NO:15. More preferably still, such a polypeptide is encodedby a nucleic acid segment that comprises an at least 150-basepaircontiguous sequence from SEQ ID NO:15. Exemplary polynucleotidesencoding the insecticidal polypeptide comprise an at least 300-basepaircontiguous nucleotide sequence from SEQ ID NO:15, and in one embodimentcomprises the nucleotide sequence of SEQ ID NO:15.

In another embodiment, the invention discloses and claims an isolatedCryET80 polypeptide comprising at least 17 contiguous amino acids fromSEQ ID NO:4. More preferably the polypeptide comprises at least 20 or atleast 23 contiguous amino acids from SEQ ID NO:4. Still more preferably,the polypeptide comprises at least 26 or at least 29 contiguous aminoacids from SEQ ID NO:4, and more preferably comprises at least 32 or atleast 35 contiguous amino acids from SEQ ID NO:4. In an exemplaryembodiment, the polypeptide comprises the sequence of SEQ ID NO:4. Sucha polypeptide is preferably encoded by a nucleic acid segment thatcomprises an at least 51-basepair contiguous nucleotide sequence fromSEQ ID NO:3, and more preferably is encoded by a nucleic acid segmentthat comprises an at least 60-basepair contiguous sequence from SEQ IDNO:3. More preferably still, such a polypeptide is encoded by a nucleicacid segment that comprises an at least 78-basepair contiguous sequencefrom SEQ ID NO:3. Exemplary polynucleotides encoding the insecticidalpolypeptide comprise an at least 96-basepair contiguous nucleotidesequence from SEQ ID NO:3, and in one embodiment comprises thenucleotide sequence of SEQ ID NO:3.

In annother embodiment, the invention provides an isolated CryET76polypeptide comprising at least 55 contiguous amino acids from SEQ IDNO:2. More preferably the polypeptide comprises at least 60 or at least70 contiguous amino acids from SEQ ID NO:2. Still more preferably, thepolypeptide comprises at least 75 or at least 80 contiguous amino acidsfrom SEQ ID NO:2, and more preferably comprises at least 85 or at least90 contiguous amino acids from SEQ ID NO:2. In an exemplary embodiment,the polypeptide comprises the sequence of SEQ ID NO:2. Such apolypeptide is preferably encoded by a nucleic acid segment thatcomprises an at least 165-basepair contiguous nucleotide sequence fromSEQ ID NO:1, and more preferably is encoded by a nucleic acid segmentthat comprises an at least 180-basepair contiguous sequence from SEQ IDNO:1. More preferably still, such a polypeptide is encoded by a nucleicacid segment that comprises an at least 225-basepair contiguous sequencefrom SEQ ID NO:1. Exemplary polynucleotides encoding the insecticidalpolypeptide comprise an at least 270-basepair contiguous nucleotidesequence from SEQ ID NO:1, and in one embodiment comprises thenucleotide sequence of SEQ ID NO:1.

In a further embodiment, the invention discloses and claims an isolatedCryET71 polypeptide comprising at least 146 contiguous amino acids fromSEQ ID NO:12. More preferably the polypeptide comprises at least 150 orat least 154 contiguous amino acids from SEQ ID NO:12. Still morepreferably, the polypeptide comprises at least 158 or at least 162contiguous amino acids from SEQ ID NO:12, and more preferably comprisesat least 166 or at least 170 contiguous amino acids from SEQ ID NO:12.In an exemplary embodiment, the polypeptide comprises the sequence ofSEQ ID NO:12. Such a polypeptide is preferably encoded by a nucleic acidsegment that comprises an at least 438-basepair contiguous nucleotidesequence from SEQ ID NO:11, and more preferably is encoded by a nucleicacid segment that comprises an at least 450-basepair contiguous sequencefrom SEQ ID NO:11. More preferably still, such a polypeptide is encodedby a nucleic acid segment that comprises at least a 462-basepaircontiguous sequence from SEQ ID NO:11. Exemplary polynucleotidesencoding the insecticidal polypeptide comprise an at least 510-basepaircontiguous nucleotide sequence from SEQ ID NO:11, and in one embodimentcomprises the nucleotide sequence of SEQ ID NO:11.

The invention also provides an isolated CryET74 polypeptide thatcomprises the sequence of SEQ ID NO:6. Such a polypeptide is preferablyencoded by a nucleic acid segment that comprises at least 45-basepaircontiguous nucleotide sequence from SEQ ID NO:5, and more preferably isencoded by a nucleic acid segment that comprises an at least 90-basepaircontiguous sequence from SEQ ID NO:5. More preferably still, such apolypeptide is encoded by a nucleic acid segment that comprises an atleast 150-basepair contiguous sequence from SEQ ID NO:5. Exemplarypolynucleotides encoding the insecticidal polypeptide comprise an atleast 300-basepair contiguous nucleotide sequence from SEQ ID NO:5, andin one embodiment comprises the nucleotide sequence of SEQ ID NO:5.

Furthermore, the invention provides an isolated CryET39 polypeptide thatcomprises the sequence of SEQ ID NO:8. Such a polypeptide is preferablyencoded by a nucleic acid segment that comprises an at least 45-basepaircontiguous nucleotide sequence from SEQ ID NO:7, and more preferably isencoded by a nucleic acid segment that comprises an at least 90-basepaircontiguous sequence from SEQ ID NO:7. More preferably still, such apolypeptide is encoded by a nucleic acid segment that comprises an atleast 150-basepair contiguous sequence from SEQ ID NO:7. Exemplarypolynucleotides encoding the insecticidal polypeptide comprise an atleast 300-basepair contiguous nucleotide sequence from SEQ ID NO:7, andin one embodiment comprises the nucleotide sequence of SEQ ID NO:7.

Likewise, the invention provides an isolated CryET79 polypeptide thatcomprises the sequence of SEQ ID NO:10. Such a polypeptide is preferablyencoded by a nucleic acid segment that comprises an at least 45-basepaircontiguous nucleotide sequence from SEQ ID NO:9, and more preferably isencoded by a nucleic acid segment that comprises an at least 90-basepaircontiguous sequence from SEQ ID NO:9. More preferably still, such apolypeptide is encoded by a nucleic acid segment that comprises an atleast 150-basepair contiguous sequence from SEQ ID NO:9. Exemplarypolynucleotides encoding the insecticidal polypeptide comprise at least300-basepair contiguous nucleotide sequence from SEQ ID NO:9, and in oneembodiment comprises the nucleotide sequence of SEQ ID NO:9.

The invention also discloses compositions and insecticidal formulationsthat comprise one or more of the polypeptides disclosed herein. Suchcomposition may be a cell extract, cell suspension, cell homogenate,cell lysate, cell supernatant, cell filtrate, or cell pellet of abacteria cell that comprises polynucleotides encoding such polypeptides.Exemplary bacterial cells that produce such polypeptides include B.thuringiensis EG4550 (deposited with the NRRL on May 30, 1997 as NRRLB-21784); EG5899 (deposited with the NRRL on May 30, 1997 as NRRLB-21783); EG11529 (deposited with the NRRL on Feb. 12, 1998 as NRRLB-21917); EG4100 (deposited with the NRRL on May 30, 1997 as NRRLB-21786); EG11647 (deposited with the NRRL on May 30, 1997 as NRRLB-21787); EG9444 (deposited with the NRRL on May 30, 1997 as NRRLB-21785); EG11648 (deposited with the NRRL on May 30, 1997 as NRRLB-21788); EG4851 (deposited with the NRRL on Feb. 12, 1998 as NRRLB-21915); and EG11658 (deposited with the NRRL on Feb. 12, 1998 as NRRLB-21916).

The composition as described in detail hereinbelow in this disclosuremay be formulated as a powder, dust, pellet, granule, spray, emulsion,colloid, solution, or such like, and may be preparable by suchconventional means as desiccation, lyophilization, homogenization,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. Preferably suchcompositions are obtainable from one or more cultures of the B.thuringiensis cells described herein. In all such compositions thatcontain at least one such insecticidal polypeptide, the polypeptide maybe present in a concentration of from about 1% to about 99% by weight.

An exemplary insecticidal polypeptide formulation may be prepared by aprocess comprising the steps of culturing a suitable B. thuringiensiscell under conditions effective to produce the insecticidalpolypeptide(s); and obtaining the insecticidal polypeptide(s) soproduced.

For example, the invention discloses and claims a method of preparing aδ-endotoxin polypeptide having insecticidal activity against acoleopteran or lepidopteran insect. The method generally involvesisolating from a suitable culture of B. thuringiensis cells that havebeen grown under appropriate conditions, one or more of the δ-endotoxinpolypeptides produced by the cells. Such polypeptides may be isolatedfrom the cell culture or supernatant or from spore suspensions derivedfrom the cell culture and used in the native form, or may be otherwisepurified or concentrated as appropriate for the particular application.

A method of controlling an insect population is also provided by theinvention. The method generally involves contacting the population withan insecticidally-effective amount of a polypeptide comprising the aminoacid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, or 19. Suchmethods may be used to kill or reduce the numbers of target insects in agiven area, or may be prophylactically applied to an environmental areato prevent infestation by a susceptible insect. Preferably the insectingests, or is contacted with, an insecticidally-effective amount of thepolypeptides.

Additionally, the invention provides a purified antibody thatspecifically binds to the insecticidal polypeptides disclosed herein.Also provided are methods of preparing such an antibody, and methods forusing the antibody to isolate, identify, characterize, and/or purifypolypeptides to which such an antibody specifically binds. Immunologicalkits and immunodetection methods useful in the identification of suchpolypeptides and peptide fragments and/or epitopes thereof are providedin detail herein, and also represent important aspects of the presentinvention.

Such antibodies may be used to detect the presence of such polypeptidesin a sample, or may be used as described hereinbelow in a variety ofimmunological methods. An exemplary method for detecting a δ-endotoxinpolypeptide in a biological sample generally involves obtaining abiological sample suspected of containing a δ-endotoxin polypeptide;contacting the sample with an antibody that specifically binds to thepolypeptide, under conditions effective to allow the formation ofcomplexes; and detecting the complexes so formed.

For such methods, the invention also provides an immunodetection kit.Such a kit generally contains, in suitable container means, an antibodythat binds to the δ-endotoxin polypeptide, and at least a firstimmunodetection reagent. Optionally, the kit may provide additionalreagents or instructions for using the antibody in the detection ofδ-endotoxin polypeptides in a sample.

Preparation of such antibodies may be achieved using the disclosedpolypeptide as an antigen in an animal as described below. Antigenicepitopes, shorter peptides, peptide fusions, carrier-linked peptidefragments, and the like may also be generated from a whole or a portionof the polypeptide sequence disclosed herein.

Another aspect of the invention relates to a biologically-pure cultureof a B. thuringiensis bacterium as shown in Table 9, deposited with theAgricultural Research Culture Collection, Northern Regional ResearchLaboratory (NRRL).

A further embodiment of the invention relates to a vector comprising asequence region that encodes a polypeptide comprising one or more of theamino acid sequences disclosed herein, a recombinant host celltransformed with such a recombinant vector, and biologically-purecultures of recombinant bacteria transformed with a polynucleotidesequence that encodes the polypeptide disclosed herein. All strainsdeposited with the NRRL were submitted to the Patent Culture Collectionunder the terms of the Budapest Treaty, and viability statementspursuant to International Receipt Form BP/4 were obtained. Exemplaryvectors, recombinant host cells, transgenic cell lines, pluripotentplant cells, and transgenic plants comprising at least a first sequenceregion that encodes a polypeptide comprising one or more of thesequences disclosed herein are described in detail hereinbelow.

In a further embodiment, the invention provides methods for preparing aninsecticidal polypeptide composition. In exemplary embodiments, suchpolypeptides may be formulated for use as an insecticidal agent, and maybe used to control insect populations in an environment, includingagricultural environs and the like. The formulations may be used to killan insect, either by topical application, or by ingestion of thepolypeptide composition by the insect. In certain instances, it may bedesirable to formulate the polypeptides of the present invention forapplication to the soil, on or near plants, trees, shrubs, and the like,near live plants, livestock, domiciles, farm equipment, buildings, andthe like.

The present invention also provides transformed host cells, pluripotentplant cell populations, embryonic plant tissue, plant calli, plantlets,and transgenic plants that comprise a seleceted sequence region thatencodes the insecticidal polypeptide. Such cells are preferablypreferably prokaryotic or eukaryotic cells such as bacterial, fungal, orplant cells, with exemplary bacterial cells including B. thuringiensis,B. subtilis, B. megaterium, B. cereus, Escherichia, Salmonella,Agrobacterium or Pseudomonas cells.

The plants and plant host cells are preferably monocotyledonous ordicotyledonous plant cells such as corn, wheat, soybean, oat, cotton,rice, rye, sorghum, sugarcane, tomato, tobacco, kapok, flax, potato,barley, turf grass, pasture grass, berry, fruit, legume, vegetable,ornamental plant, shrub, cactus, succulent, and tree cell.

Illustrative transgenic plants of the present invention preferably haveincorporated into their genome a selected polynucleotide (or“transgene”), that comprises at least a first sequence region thatencodes one or more of the insecticidal polypeptides disclosed herein.

Likewise, a progeny (decendant, offspring, etc.) of any generation ofsuch a transgenic plant also represents an important aspect of theinvention. Preferably such progeny comprise the selected transgene, andinherit the phenotypic trait of insect resistance demonstrated by theparental plant. A seed of any generation of all such transgenicinsect-resistant plants is also an important aspect of the invention.Preferably the seed will also comprise the selected transgene and willconfer to the plants grown from the seed the phenotypic trait of insectresistance.

Insect resistant, crossed fertile transgenic plants comprising one ormore transgenes that encode one or more of the polypeptides disclosedherein may be prepared by a method that generally involves obtaining afertile transgenic plant that contains a chromosomally incorporatedtransgene encoding such an insecticidal polypeptide; operably linked toa promoter active in the plant; crossing the fertile transgenic plantwith a second plant lacking the transgene to obtain a third plantcomprising the transgene; and backcrossing the third plant to obtain abackcrossed fertile plant. In such cases, the transgene may be inheritedthrough a male parent or through a female parent. The second plant maybe an inbred, and the third plant may be a hybrid.

Likewise, an insect resistant hybrid, transgenic plant may be preparedby a method that generally involves crossing a first and a second inbredplant, wherein one or both of the first and second inbred plantscomprises a chromosomally incorporated transgene that encodes theselected polypeptide operably linked to a plant expressible promoterthat expresses the transgene. In illustrative embodiments, the first andsecond inbred plants may be monocot plants selected from the groupconsisting of: corn, wheat, rice, barley, oats, rye, sorghum, turfgrassand sugarcane.

In related embodiment, the invention also provides a method of preparingan insect resistant plant. The method generally involves contacting arecipient plant cell with a DNA composition comprising at least a firsttransgene that encodes an insecticidal polypeptide under conditionspermitting the uptake of the DNA composition; selecting a recipient cellcomprising a chromosomally incorporated transgene that encodes thepolypeptide; regenerating a plant from the selected cell; andidentifying a fertile transgenic plant that has enhanced insectresistance relative to the corresponding non-transformed plant.

A method of producing transgenic seed generally involves obtaining afertile transgenic plant comprising a chromosomally integrated transgenethat encodes a polypeptide comprising one or more of the amino acidsequences disclosed herein, operably linked to a promoter that expressesthe transgene in a plant; and growing the plant under appropriateconditions to produce the transgenic seed.

A method of producing progeny of any generation of an insectresistance-enhanced fertile transgenic plant is also provided by theinvention. The method generally involves collecting transgenic seed froma transgenic plant comprising a chromosomally integrated transgene thatencodes such a polypeptide, operably linked to a promoter that expressesthe transgene in the plant; planting the collected transgenic seed; andgrowing the progeny transgenic plants from the seed.

These methods for creating transgenic plants, progeny and seed mayinvolve contacting the plant cell with the DNA composition using one ofthe processes well-known for plant cell transformation such asmicroprojectile bombardment, electroporation or Agrobacterium-mediatedtransformation. These and other embodiments of the present inventionwill be apparent to those of skill in the art from the followingexamples and claims, having benefit of the teachings of the Specficationherein.

2.1 Polynucleotide Segments

The present invention provides nucleic acid segments, that can beisolated from virtually any source, that are free from total genomic DNAand that encode the novel insecticidal polypeptides and peptidefragments thereof that are disclosed herein. The polynucleotidesencoding these peptides and polypeptides may encode active insecticidalproteins, or peptide fragments, polypeptide subunits, functionaldomains, or the like of one or more of the CryET84, CryET80, CryET76,CryET71, CryET69, CryET75, CryET39, CryET79, CryET74 and related crystalproteins as the polypeptides disclosed herein. In addition the inventionencompasses nucleic acid segments which may be synthesized entirely invitro using methods that are well-known to those of skill in the artwhich encode the novel polypeptides, peptides, peptide fragments,subunits, or functional domains disclosed herein.

As used herein, the term “nucleic acid segment” or “polynucleotide”refers to a nucleic acid molecule that has been isolated free of thetotal genomic DNAs of a particular species. Therefore, a nucleic acidsegment or polynucleotide encoding an endotoxin polypeptide refers to anucleic acid molecule that comprises at least a first crystalprotein-encoding sequences yet is isolated away from, or purified freefrom, total genomic DNA of the species from which the nucleic acidsegment is obtained, which in the instant case is the genome of theGram-positive bacterial genus, Bacillus, and in particular, the speciesof Bacillus known as B. thuringiensis. Included within the term “nucleicacid segment”, are polynucleotide segments and smaller fragments of suchsegments, and also recombinant vectors, including, for example,plasmids, cosmids, phagemids, phage, virions, baculoviruses, artificialchromosomes, viruses, and the like. Accordingly, polynucleotidesequences that have between about 70% and about 80%, or more preferablybetween about 81% and about 90%, or even more preferably between about91% and about 99% nucleic acid sequence identity or functionalequivalence to the polynucleotide sequence of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQID NO:15, or SEQ ID NO:18 will be sequences that are “essentially as setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:18.” Highlypreferred sequences, are those which are preferably about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100% identical or functionally equivalent to thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:13, SEQ IDNO:15, or SEQ ID NO:18. Other preferred sequences that encode relatedpolypeptide sequences are those which are about 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, or 90% identical or functionally equivalent to thepolynucleotide sequence set forth in one or more of these sequenceidentifiers. Likewise, sequences that are about 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, or 80% identical or functionally equivalent to thepolynucleotide sequence set forth in one or more of these sequenceidentifiers are also contemplated to be useful in the practice of thepresent invention.

Similarly, a polynucleotide comprising an isolated, purified, orselected gene or sequence region refers to a polynucleotide which mayinclude in addition to peptide encoding sequences, certain otherelements such as, regulatory sequences, isolated substantially away fromother naturally occurring genes or protein-encoding sequences. In thisrespect, the term “gene” is used for simplicity to refer to a functionalprotein-, or polypeptide-encoding unit. As will be understood by thosein the art, this functional term includes both genomic sequences,operator sequences and smaller engineered gene segments that express, ormay be adapted to express, proteins, polypeptides or peptides. Incertain embodiments, a nucleic acid segment will comprise at least afirst gene that encodes one or more of the polypeptides disclosedherein.

To permit expression of the gene, and translation of the mRNA intomature polypeptide, the nucleic acid segment preferably also comprisesat least a first promoter operably linked to the gene to express thegene product in a host cell transformed with this nucleic acid segment.The promoter may be an endogenous promoter, or alternatively, aheterologous promoter selected for its ability to promote expression ofthe gene in one or more particular cell types. For example, in thecreation of transgenic plants and pluripotent plant cells comprising aselected gene, the heterologous promoter of choice is one that isplant-expressible, and in many instances, may preferably be aplant-expressible promoter that is tissue- or cell cycle-specific. Theselection of plant-expressible promoters is well-known to those skilledin the art of plant transformation, and exemplary suitable promoters aredescribed herein. In certain embodiments, the plant-expressible promotermay be selected from the group consisting of corn sucrose synthetase 1,corn alcohol dehydrogenase 1, corn light harvesting complex, corn heatshock protein, pea small subunit RuBP carboxylase, Ti plasmid mannopinesynthase, Ti plasmid nopaline synthase, petunia chalcone isomerase, beanglycine rich protein 1, Potato patatin, lectin, CaMV 35S, and the S-E9small subunit RuBP carboxylase promoter.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a gene encoding a bacterial crystalprotein, forms the significant part of the coding region of the DNAsegment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or operon coding regions. Of course, this refersto the DNA segment as originally isolated, and does not exclude genes,recombinant genes, synthetic linkers, or coding regions later added tothe segment by the hand of man.

In particular embodiments, the invention concerns isolatedpolynucleotides (such as DNAs, RNAs, antisense DNAs, antisense RNAs,ribozymes, and PNAs) and recombinant vectors comprising polynucleotidesequences that encode one or more of the polypeptides disclosed herein.

The term “a sequence essentially as set forth in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:19” means that the sequencesubstantially corresponds to a portion of the sequence of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, or SEQ ID NO:19 and has relatively few aminoacids that are not identical to, or a biologically functional equivalentof, the amino acids of any of these sequences. The term “biologicallyfunctional equivalent” is well understood in the art and is furtherdefined in detail herein (e.g., see Illustrative Embodiments).Accordingly, sequences that have between about 70% and about 80%, ormore preferably between about 81% and about 90%, or even more preferablybetween about 91% and about 99% amino acid sequence identity orfunctional equivalence to the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:19 will be sequences that are“essentially as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQID NO:19.” Highly preferred sequences, are those which are preferablyabout 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100% identical or functionallyequivalent to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:14, SEQ ID NO:16, or SEQ ID NO:19. Other preferred sequences arethose which are about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or90% identical or functionally equivalent to the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:19.Likewise, sequences that are about 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, or 80% identical or functionally equivalent to the polypeptidesequence set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:14, SEQ IDNO:16, or SEQ ID NO:19 are also contemplated to be useful in thepractice of the present invention.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other nucleicacid sequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant nucleicacid protocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding the peptide sequencedisclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:19, orthat are identical to or complementary to nucleic acid sequences whichencode the peptides disclosed in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, or SEQ ID NO:16,or SEQ ID NO:19, and particularly those nucleic acid segments disclosedin SEQ ID NOS:1, 3, 13, 15, or 18. For example, nucleic acid sequencessuch as about 23 nucleotides, and that are up to about 10,000, about5,000, about 3,000, about 2,000, about 1,000, about 500, about 200,about 100, about 50, and about 23 or so base pairs in length (includingall intermediate lengths) that comprise a contiguous nucleotide sequencefrom SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:18 or those thatencode a contiguous amino acid sequence from SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, or SEQ ID NO:19 are contemplated to be particularly useful.

It will be readily understood that “intermediate lengths”, in thecontext of polynucleotide sequences, or nucleic acid segments, or primeror probes specific for the disclosed gene, means any length between thequoted ranges, such as from about 24, 25, 26, 27, 28, 29, etc.; 30, 31,32, 33, 34, 35, 36, 37, 38, 39, etc.; 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85,90, 95, etc.; 100, 101, 102, 103, 104, etc.; 110, 120, 125, 130, 135,140, 145, 150, 155, 160, 165, 170, 180, 190, etc.; including allintegers in the ranges of from about 200-500; 500-1,000; 1,000-2,000;2,000-3,000; 3,000-5,000; and up to and including sequences of about10,000 or 12,000 or so nucleotides and the like.

Likewise, it will be readily understood that “intermediate lengths”, inthe context of polypeptides or peptides, means any length between thequoted ranges of contiguous amino acids. For example, when consideringthe disclosed insecticidal polypeptides, all lengths between about 7 andabout 300 contiguous amino acid sequences are contemplated to be usefulin particular embodiments disclosed herein. For example, peptidescomprising contiguous amino acid sequences having about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 65, etc., 70, 75, etc., 80, 85, etc., 90, 95, etc., and eventhose peptides comprising at least about 96, 97, 98, 99, 100, 101, 102,103, and 104, or more contigous amino acids from SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:19 are explicitly considered to fallwithin the scope of the present invention.

Furthermore, it will also be readily understood by one of skill in theart, that “intermediate lengths”, in the context of largerinsecticidally-active polypeptides, means any length between the quotedranges of contiguous amino acids that comprise such a polypeptide. Forexample, when considering the polypeptides of the present invention, alllengths between about 100 and about 1000 contiguous amino acid sequencesare contemplated to be useful in particular embodiments disclosedherein. For example, polypeptides comprising a contiguous amino acidsequence having at least about 100, about 101, about 102, 103, 104, 105,106, 107, 108, 109, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,160, 165, 170, 175, 180, 185, 190, 195, etc., 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 220, 230, 240, 250, 260, 270, 280, 290,etc., 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, etc., 410,430, 450, 470, 490, etc., 500, 525, 550, 575, 600, 650, 675, 700, etc.,750, etc., and even those polypeptides that comprise at least about 775or more amino acids are explicitly considered to fall within the scopeof the present invention. Particularly in the case of fusion proteinscomprising a whole or a portion of the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:19 longer polypeptide sequencesmay be preferred, including sequences that comprise about 760, 770, 780,790, or even about 800 or 900 or greater amino acids in length.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention, or which encode the amino acid sequence of SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:19 including the DNA sequence which isparticularly disclosed in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ IDNO:18. Recombinant vectors and isolated DNA segments may thereforevariously include the polypeptide-coding regions themselves, codingregions bearing selected alterations or modifications in the basiccoding region, or they may encode larger polypeptides that neverthelessinclude these peptide-coding regions or may encode biologicallyfunctional equivalent proteins or peptides that have variant amino acidssequences.

The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon degeneracy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level. Alternatively, native,as yet-unknown or as yet unidentified polynucleotides and/orpolypeptides structurally and/or functionally-related to the sequencesdisclosed herein may also be identified that fall within the scope ofthe present invention. Such polynucleotides are those polynucleotidesthat encode a polypeptide structurally and/or functionally similar oridentical to, the polypeptide characterized herein as a crystalprotein-encoding polynucleotide. Since the designations “CryET39,”“CryET69,” “CryET71,” “CryET74,” “CryET76,” “CryET79,” “CryEt80,”“CryET84” and “CryET75” are arbitrary names chosen to readily identifypolypeptides comprising the amino acid sequences disclosed herein, it islikely that many other polypeptides may be identified that are highlyhomologous to (or even identical to) this sequence, but which may havebeen isolated from different organisms or sources, or alternatively, mayeven have been synthesized entirely, or partially de novo. As such, allpolypeptide sequences, whether naturally-occurring, orartificially-created, that are structurally homologous to the primaryamino acid sequences as described herein and that have similarinsecticidal activity against the target insects disclosed herein areconsidered to fall within the scope of this disclosure. Likewise, allpolynucleotide sequences, whether naturally-occurring, orartificially-created, that are structurally homologous to the nucleotidesequences disclosed herein, or that encodes a polypeptide that ishomologous, and biologically-functionally equivalent to the amino acidsequence disclosed herein are also considered to fall within the scopeof this disclosure.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding afull-length insecticidal protein or smaller peptide, is positioned underthe control of a promoter. The promoter may be in the form of thepromoter that is naturally associated with a gene encoding peptides ofthe present invention, as may be obtained by isolating the 5′ non-codingsequences located upstream of the coding segment or exon, for example,using recombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein. In many cases, the promoter may be anative promoter, or alternatively, a heterologous promoter, such asthose of bacterial origin (including promoters from other crystalproteins), fungal origin, viral, phage or phagemid origin (includingpromoters such as CaMV35, and its derivatives, T3, T7, λ, and φpromoters and the like), or plant origin (including constitutive,inducible, and/or tissue-specific promoters and the like).

2.1.1 Characteristics of the CryET76, CryET80 and CryET84 PolypeptidesIsolated from EG4851

The present invention provides a novel polypeptide that defines a wholeor a portion of a B. thuringiensis CryET76, CryET84 or a CryET80 crystalproteins.

In a preferred embodiment, the invention discloses and claims anisolated and purified CryET76 protein. The CryET76 protein isolated fromEG4851 comprises a 387-amino acid sequence, and has a calculatedmolecular mass of approximately 43,800 Da. CryET76 has a calculatedisoelectric constant (pI) equal to 5.39.

In a preferred embodiment, the invention discloses and claims anisolated and purified CryET80 protein. The CryET80 protein isolated fromEG4851 comprises a 132-amino acid sequence, and has a calculatedmolecular mass of approximately 14,800 Da. CryET80 has a calculatedisoelectric constant (pI) equal to 6.03.

In a preferred embodiment, the invention discloses and claims anisolated and purified CryET84 protein. The CryET84 protein isolated fromEG4851 comprises a 341-amino acid sequence, and has a calculatedmolecular mass of approximately 37,884 Da. CryET84 has a calculatedisoelectric constant (pI) equal to 5.5.

In strain EG4851, the cryET80 and cryET76 genes are preferably locatedon a single DNA segment and are separated by about 95 nucleotides. Thegene for CryET76 extends from nucleotide nucleotide 514 to nucleotide1674 of SEQ ID NO:5, and the gene encoding CryET80 extends fromnucleotide 23 to nucleotide 418 of SEQ ID NO:5. In the presentinvention, the cryET80 and cryET76 genes may be preferably located on asingle DNA segment.

In strain EG4851, the cryET84 gene is located immediately 5′ to thecryET80 and cryET76 genes. The nucleotide sequence of the cryET84 geneis shown in SEQ ID NO:18 and the deduced amino acid sequence of theCryET84 protein is shown in SEQ ID NO:19. In the present invention, thecryET80, cryET84, and cryET76 genes may be preferably located on asingle DNA segment (e.g. SEQ ID NO:17).

2.2 Nucleic Acid Segments as Hybridization Probes and Primers

In addition to their use in directing the expression of crystal proteinsor peptides of the present invention, the nucleic acid sequencesdescribed herein also have a variety of other uses. For example, theyhave utility as probes or primers in nucleic acid hybridizationembodiments. The invention provides a method for detecting a nucleicacid sequence encoding a δ-endotoxin polypeptide. The method generallyinvolves obtaining sample nucleic acids suspected of encoding aδ-endotoxin polypeptide; contacting the sample nucleic acids with anisolated nucleic acid segment comprising one of the sequences disclosedherein, under conditions effective to allow hybridization ofsubstantially complementary nucleic acids; and detecting the hybridizedcomplementary nucleic acids thus formed.

Also provided is a nucleic acid detection kit comprising, in suitablecontainer means, at least a first nucleic acid segment comprising atleast 23 contiguous nucleotides from SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15or SEQ ID NO:18, and at least a first detection reagent. The ability ofsuch nucleic acid probes to specifically hybridize to crystalprotein-encoding sequences will enable them to be of use in detectingthe presence of complementary sequences in a given sample. However,other uses are envisioned, including the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of about 23 to about 50, or even up to andincluding sequences of about 100-200 nucleotides or so, identical orcomplementary to the DNA sequences herein, are particularly contemplatedas hybridization probes for use in, e.g., Southern and Northernblotting. Intermediate-sized fragments will also generally find use inhybridization embodiments, wherein the length of the contiguouscomplementary region may be varied, such as between about 25-30, orbetween about 30 and about 40 or so nucleotides, but larger contiguouscomplementary stretches may be used, such as those from about 200 toabout 300, or from about 300 to about 400 or 500 or so nucleotides inlength, according to the length complementary sequences one wishes todetect. It is even possible that longer contiguous sequence regions maybe utilized including those sequences comprising at least about 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or more contiguousnucleotides from one of the sequences disclosed herein.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195 and4,683,202 (each incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids. “Highstringency” hybridization conditions, e.g., typically employ relativelylow salt and/or high temperature conditions, such as provided by about0.02 M to about 0.15 M NaCl at temperatures of about 50° C. to about 70°C. Such selective conditions tolerate little, if any, mismatch betweenthe probe and the template or target strand, and would be particularlysuitable for isolating crystal protein-encoding DNA segments. Detectionof DNA segments via hybridization is well-known to those of skill in theart, and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (eachincorporated herein by reference) are exemplary of the methods ofhybridization analyses. Teachings such as those found in the texts ofMaloy et al., 1990; Maloy 1994; Segal, 1976; Prokop and Bajpai, 1991;and Kuby, 1994, are particularly relevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ “low stringency” or “reducedstringency” hybridization conditions such as those employing from about0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. toabout 55° C. Cross-hybridizing species can thereby be readily identifiedas positively hybridizing signals with respect to controlhybridizations. In any case, it is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts offormamide, which serves to destabilize the hybrid duplex in the samemanner as increased temperature. Thus, hybridization conditions can bereadily manipulated, and thus will generally be a method of choicedepending on the desired results. Regardless of what particularcombination of salts (such as NaCl or NaCitrate and the like), organicbuffers (including e.g., formamide and the like), and incubation orwashing temperatures are employed, the skilled artisan will readily beable to employ hybridization conditions that are “high,” “medium,” or“low” stringency, and will be able to interpret the results fromhybridization analyses using such conditions to determine the relativehomology of a target nucleic acid sequence to that of the particularnovel polynucleotide probe sequence employed from SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15 or SEQ ID NO:18.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmentally undesirable reagents. In the case of enzyme tags,calorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

2.3 Vectors and Methods for Recombinant Expression of Cry RelatedPolypeptides

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a crystal protein orpeptide in its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or plant cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type, organism, or even animal, chosenfor expression. The use of promoter and cell type combinations forprotein expression is generally known to those of skill in the art ofmolecular biology, for example, see Sambrook et al., (1989). Thepromoters employed may be constitutive, or inducible, and can be usedunder the appropriate conditions to direct high level expression of theintroduced DNA segment, such as is advantageous in the large-scaleproduction of recombinant proteins or peptides. Appropriate promotersystems contemplated for use in high-level expression include, but arenot limited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8 to about 50 aminoacids in length, or more preferably, from about 8 to about 30 aminoacids in length, or even more preferably, from about 8 to about 20 aminoacids in length are contemplated to be particularly useful. Such peptideepitopes may be amino acid sequences which comprise a contiguous aminoacid sequence as disclosed herein.

2.4 Transgenic Plants Expressing CryET Polypeptides

In yet another aspect, the present invention provides methods forproducing a transgenic plant that expresses a selected nucleic acidsegment comprising a sequence region that encodes the novel endotoxinpolypeptides of the present invention. The process of producingtransgenic plants is well-known in the art. In general, the methodcomprises transforming a suitable plant host cell with a DNA segmentthat contains a promoter operatively linked to a coding region thatencodes one or more of the disclosed polypeptides. Such a coding regionis generally operatively linked to at least a firsttranscription-terminating region, whereby the promoter is capable ofdriving the transcription of the coding region in the cell, and henceproviding the cell the ability to produce the polypeptide in vivo.Alternatively, in instances where it is desirable to control, regulate,or decrease the amount of a particular recombinant crystal proteinexpressed in a particular transgenic cell, the invention also providesfor the expression of crystal protein antisense mRNA. The use ofantisense mRNA as a means of controlling or decreasing the amount of agiven protein of interest in a cell is well-known in the art.

Another aspect of the invention comprises transgenic plants whichexpress a gene, gene segment, or sequence region that encodes at leastone or more of the novel polypeptide compositions disclosed herein. Asused herein, the term “transgenic plant” is intended to refer to a plantthat has incorporated DNA sequences, including but not limited to geneswhich are perhaps not normally present, DNA sequences not normallytranscribed into RNA or translated into a protein (“expressed”), or anyother genes or DNA sequences which one desires to introduce into thenon-transformed plant, such as genes which may normally be present inthe non-transformed plant but which one desires to either geneticallyengineer or to have altered expression.

It is contemplated that in some instances the genome of a transgenicplant of the present invention will have been augmented through thestable introduction of one or more transgenes, either native,synthetically modified, or mutated, that encodes an insecticidalpolypeptide that is identical to, or highly homologous to thepolypeptide disclosed herein. In some instances, more than one transgenewill be incorporated into the genome of the transformed host plant cell.Such is the case when more than one crystal protein-encoding DNA segmentis incorporated into the genome of such a plant. In certain situations,it may be desirable to have one, two, three, four, or even more B.thuringiensis crystal proteins (either native orrecombinantly-engineered) incorporated and stably expressed in thetransformed transgenic plant. Alternatively, a second transgene may beintroduced into the plant cell to confer additional phenotypic traits tothe plant. Such transgenes may confer resistance to one or more insects,bacteria, fungi, viruses, nematodes, or other pathogens.

A preferred gene which may be introduced includes, for example, acrystal protein-encoding DNA sequence from bacterial origin, andparticularly one or more of those described herein which are obtainedfrom Bacillus spp. Highly preferred nucleic acid sequences are thoseobtained from B. thuringiensis, or any of those sequences which havebeen genetically engineered to decrease or increase the insecticidalactivity of the crystal protein in such a transformed host cell.

Means for transforming a plant cell and the preparation of pluripotentplant cells, and regeneration of a transgenic cell line from atransformed cell, cell culture, embryo, or callus tissue are well-knownin the art, and are discussed herein. Vectors, (including plasmids,cosmids, phage, phagemids, baculovirus, viruses, virions, BACs[bacterial artificial chromosomes], YACs [yeast artificial chromosomes])comprising at least a first nucleic acid segment encoding aninsecticidal polypeptide for use in transforming such cells will, ofcourse, generally comprise either the operons, genes, or gene-derivedsequences of the present invention, either native, orsynthetically-derived, and particularly those encoding the disclosedcrystal proteins. These nucleic acid constructs can further includestructures such as promoters, enhancers, polylinkers, introns,terminators, or even gene sequences which have positively- ornegatively-regulating activity upon the cloned δ-endotoxin gene asdesired. The DNA segment or gene may encode either a native or modifiedcrystal protein, which will be expressed in the resultant recombinantcells, and/or which will confer to a transgenic plant comprising such asegment, an improved phenotype (in this case, increased resistance toinsect attack, infestation, or colonization).

The preparation of a transgenic plant that comprises at least onepolynucleotide sequence encoding an insecticidal polypeptide for thepurpose of increasing or enhancing the resistance of such a plant toattack by a target insect represents an important aspect of theinvention. In particular, the inventors describe herein the preparationof insect-resistant monocotyledonous or dicotyledonous plants, byincorporating into such a plant, a transgenic DNA segment encoding oneor more insecticidal polypeptides which are toxic to a coleopteran orlepidopteran insect.

In a related aspect, the present invention also encompasses a seedproduced by the transformed plant, a progeny from such seed, and a seedproduced by the progeny of the original transgenic plant, produced inaccordance with the above process. Such progeny and seeds will have acrystal protein-encoding transgene stably incorporated into theirgenome, and such progeny plants will inherit the traits afforded by theintroduction of a stable transgene in Mendelian fashion. All suchtransgenic plants having incorporated into their genome transgenic DNAsegments encoding one or more crystal proteins or polypeptides areaspects of this invention. As well-known to those of skill in the art, aprogeny of a plant is understood to mean any offspring or any descendantfrom such a plant.

2.5 Crystal Protein Screening and Detection Kits

The present invention contemplates methods and kits for screeningsamples suspected of containing crystal protein polypeptides or crystalprotein-related polypeptides, or cells producing such polypeptides. Akit may contain one or more antibodies specific for the disclosed aminoacid sequences disclosed, or one or more antibodies specific for apeptide derived from one of the sequences disclosed, and may alsocontain reagent(s) for detecting an interaction between a sample and anantibody of the present invention. The provided reagent(s) can beradio-, fluorescently- or enzymatically-labeled. The kit can contain aknown radiolabeled agent capable of binding or interacting with anucleic acid or antibody of the present invention.

The reagent(s) of the kit can be provided as a liquid solution, attachedto a solid support or as a dried powder. Preferably, when the reagent(s)are provided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent(s) provided are attached to asolid support, the solid support can be chromatograph media, a testplate having a plurality of wells, or a microscope slide. When thereagent(s) provided are a dry powder, the powder can be reconstituted bythe addition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thecrystal proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect crystal proteins or crystal protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a crystal protein or peptide or a crystalprotein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the tittering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of crystal proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containingcrystal proteins or peptides. Generally speaking, kits in accordancewith the present invention will include a suitable crystal protein,peptide or an antibody directed against such a protein or peptide,together with an immunodetection reagent and a means for containing theantibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

2.6 Insecticidal Compositions and Methods of Use

The inventors contemplate that the polypeptide compositions disclosedherein will find particular utility as insecticides for topical and/orsystemic application to field crops, grasses, fruits and vegetables,lawns, trees, and/or ornamental plants. Alternatively, the polypeptidesdisclosed herein may be formulated as a spray, dust, powder, or otheraqueous, atomized or aerosol for killing an insect, or controlling aninsect population. The polypeptide compositions disclosed herein may beused prophylactically, or alternatively, may be administered to anenvironment once target insects, such as WCRW, have been identified inthe particular environment to be treated. The polypeptide compositionsmay comprise an individual Cry polypeptide or may contain variouscombinations of the polypeptides disclosed herein.

Regardless of the method of application, the amount of the activepolypeptide component(s) is applied at an insecticidally-effectiveamount, which will vary depending on such factors as, for example, thespecific target insects to be controlled, the specific environment,location, plant, crop, or agricultural site to be treated, theenvironmental conditions, and the method, rate, concentration,stability, and quantity of application of the insecticidally-activepolypeptide composition. The formulations may also vary with respect toclimatic conditions, environmental considerations, and/or frequency ofapplication and/or severity of insect infestation.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in insecticide formulation technology; theseare well known to those skilled in insecticide formulation. Theformulations may be mixed with one or more solid or liquid adjuvants andprepared by various means, e.g., by homogeneously mixing, blendingand/or grinding the insecticidal composition with suitable adjuvantsusing conventional formulation techniques.

2.6.1 Oil Flowable Suspensions

In a preferred embodiment, the bioinsecticide composition comprises anoil flowable suspension of bacterial cells which expresses the novelcrystal protein disclosed herein. Exemplary bacterial species includethose such as B. thuringiensis, B. megaterium, B. subtilis, B. cereus,E. coli, Salmonella spp., Agrobacterium spp., or Pseudomonas spp.

2.6.2 Water-Dispersible Granules

In another important embodiment, the bioinsecticide compositioncomprises a water dispersible granule. This granule comprises bacterialcells which expresses a novel crystal protein disclosed herein.Preferred bacterial cells include bacteria such as B. megaterium, B.subtilis, B. cereus, E. coli, Salmonella spp., Agrobacterium spp., orPseudomonas spp. cells transformed with a DNA segment disclosed hereinand expressing the crystal protein are also contemplated to be useful.

2.6.3 Powders, Dusts, and Spore Formulations

In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, dust, spore crystal formulation, cellpellet, or colloidal concentrate. This powder comprises bacterial cellswhich expresses a novel crystal protein disclosed herein. Preferredbacterial cells include B. thuringiensis cells, or cells of strains ofbacteria such as B. megaterium, B. subtilis, B. cereus, E. coli,Salmonella spp., Agrobacterium spp., or Pseudomonas spp. and the like,may also be transformed with one or more nucleic acid segments asdisclosed herein. Such dry forms of the insecticidal compositions may beformulated to dissolve immediately upon wetting, or alternatively,dissolve in a controlled-release, sustained-release, or othertime-dependent manner. Such compositions may be applied to, or ingestedby, the target insect, and as such, may be used to control the numbersof insects, or the spread of such insects in a given environment.

2.6.4 Aqueous Suspensions and Bacterial Cell Filtrates or Lysates

In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells or an aqueoussuspension of parasporal crystals, or an aqueous suspension of bacterialcell lysates or filtrates, etc., such as those described above whichexpress the crystal protein. Such aqueous suspensions may be provided asa concentrated stock solution which is diluted prior to application, oralternatively, as a diluted solution ready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the crystal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise intact B. thuringiensiscells expressing the protein of interest, such bacteria may beformulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various inert materials, suchas inorganic minerals (phyllosilicates, carbonates, sulfates,phosphates, and the like) or botanical materials (powdered corncobs,rice hulls, walnut shells, and the like). The formulations may includespreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include Theologicalagents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel insecticidal polypeptides may be prepared bynative or recombinant bacterial expression systems in vitro and isolatedfor subsequent field application. Such protein may be either in crudecell lysates, suspensions, colloids, etc., or alternatively may bepurified, refined, buffered, and/or further processed, beforeformulating in an active biocidal formulation. Likewise, under certaincircumstances, it may be desirable to isolate crystals and/or sporesfrom bacterial cultures expressing the crystal protein and applysolutions, suspensions, or colloidal preparations of such crystalsand/or spores as the active bioinsecticidal composition.

2.6.5 Multifunctional Formulations

In certain embodiments, when the control of multiple insect species isdesired, the insecticidal formulations described herein may also furthercomprise one or more chemical pesticides, (such as chemical pesticides,nematocides, fungicides, virucides, microbicides, amoebicides,insecticides, etc.), and/or one or more δ-endotoxin polypeptides havingthe same, or different insecticidal activities or insecticidalspecificities, as the insecticidal polypeptide identified herein. Theinsecticidal polypeptides may also be used in conjunction with othertreatments such as fertilizers, weed killers, cryoprotectants,surfactants, detergents, insecticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. Likewise the formulations may be prepared into edible“baits” or fashioned into insect “traps” to permit feeding or ingestionby a target insect of the insecticidal formulation.

The insecticidal compositions of the invention may also be used inconsecutive or simultaneous application to an environmental site singlyor in combination with one or more additional insecticides, pesticides,chemicals, fertilizers, or other compounds.

2.6.6 Application Methods and Effective Rates

The insecticidal compositions of the invention are applied to theenvironment of the target insect, typically onto the foliage of theplant or crop to be protected, by conventional methods, preferably byspraying. The strength and duration of insecticidal application will beset with regard to conditions specific to the particular pest(s),crop(s) to be treated and particular environmental conditions. Theproportional ratio of active ingredient to carrier will naturally dependon the chemical nature, solubility, and stability of the insecticidalcomposition, as well as the particular formulation contemplated.

Other application techniques, including dusting, sprinkling, soilsoaking, soil injection, seed coating, seedling coating, foliarspraying, aerating, misting, atomizing, fumigating, aerosolizing, andthe like, are also feasible and may be required under certaincircumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well-known to those ofskill in the art.

The insecticidal compositions of the present invention may also beformulated for preventative or prophylactic application to an area, andmay in certain circumstances be applied to pets, livestock, animalbedding, or in and around farm equipment, barns, domiciles, oragricultural or industrial facilities, and the like.

The concentration of insecticidal composition which is used forenvironmental, systemic, topical, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 1% by weightand may be up to and including about 99% by weight. Dry formulations ofthe polypeptide compositions may be from about 1% to about 99% or moreby weight of the protein composition, while liquid formulations maygenerally comprise from about 1% to about 99% or more of the activeingredient by weight. As such, a variety of formulations are preparable,including those formulations that comprise from about 5% to about 95% ormore by weight of the insecticidal polypeptide, including thoseformulations that comprise from about 10% to about 90% or more by weightof the insecticidal polypeptide. Naturally, compositions comprising fromabout 15% to about 85% or more by weight of the insecticidalpolypeptide, and formulations comprising from about 20% to about 80% ormore by weight of the insecticidal polypeptide are also considered tofall within the scope of the present disclosure.

In the case of compositions in which intact bacterial cells that containthe insecticidal polypeptide are included, preparations will generallycontain from about 10⁴ to about 10⁸ cells/mg, although in certainembodiments it may be desirable to utilize formulations comprising fromabout 10² to about 10⁴ cells/mg, or when more concentrated formulationsare desired, compositions comprising from about 10⁸ to about 10¹⁰ or10¹¹ cells/mg may also be formulated. Alternatively, cell pastes, sporeconcentrates, or crystal protein suspension concentrates may be preparedthat contain the equivalent of from about 10¹² to 10¹³ cells/mg of theactive polypeptide, and such concentrates may be diluted prior toapplication.

The insecticidal formulation described above may be administered to aparticular plant or target area in one or more applications as needed,with a typical field application rate per hectare ranging on the orderof from about 50 g/hectare to about 500 g/hectare of active ingredient,or alternatively, from about 500 g/hectare to about 1000 g/hectare maybe utilized. In certain instances, it may even be desirable to apply theinsecticidal formulation to a target area at an application rate of fromabout 1000 g/hectare to about 5000 g/hectare or more of activeingredient. In fact, all application rates in the range of from about 50g of active polypeptide per hectare to about 10,000 g/hectare arecontemplated to be useful in the management, control, and killing, oftarget insect pests using such insecticidal formulations. As such, ratesof about 100 g/hectare, about 200 g/hectare, about 300 g/hectare, about400 g/hectare, about 500 g/hectare, about 600 g/hectare, about 700g/hectare, about 800 g/hectare, about 900 g/hectare, about 1 kg/hectare,about 1.1 kg/hectare, about 1.2 kg/hectare, about 1.3 kg/hectare, about1.4 kg/hectare, about 1.5 kg/hectare, about 1.6 kg/hectare, about 1.7kg/hectare, about 1.8 kg/hectare, about 1.9 kg/hectare, about 2.0kg/hectare, about 2.5 kg/hectare, about 3.0 kg/hectare, about 3.5kg/hectare, about 4.0 kg/hectare, about 4.5 kg/hectare, about 6.0kg/hectare, about 7.0 kg/hectare, about 8.0 kg/hectare, about 8.5kg/hectare, about 9.0kg/hectare, and even up to and including about 10.0kg/hectare or greater of active polypeptide may be utilized in certainagricultural, industrial, and domestic applications of the pesticidalformulations described hereinabove.

2.7 Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified peptide which incorporates an epitope that is immunologicallycross-reactive with one or more antibodies that are specific for thedisclosed polypeptide sequences. In particular, the invention concernsepitopic core sequences derived from one or more of the polypeptidesdisclosed herein.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more antibodies that arespecific for the disclosed polypeptide sequence” is intended to refer toa peptide or protein antigen which includes a primary, secondary ortertiary structure similar to an epitope located within the disclosedpolypeptide. The level of similarity will generally be to such a degreethat monoclonal or polyclonal antibodies directed against the crystalprotein or polypeptide will also bind to, react with, or otherwiserecognize, the cross-reactive peptide or protein antigen. Variousimmunoassay methods may be employed in conjunction with such antibodies,such as, for example, Western blotting, ELISA, RIA, and the like, all ofwhich are known to those of skill in the art.

The identification of immunodominant epitopes, and/or their functionalequivalents, suitable for use in vaccines is a relativelystraightforward matter. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, for example, Jameson andWolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acidsequence of these “epitopic core sequences” may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of about 8 to about 20 amino acids in length,and more preferably about 8 to about 15 amino acids in length. It isproposed that shorter antigenic crystal protein-derived peptides willprovide advantages in certain circumstances, for example, in thepreparation of immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to crystal proteinsand related sequences. These epitopic core sequences are identifiedherein in particular aspects as hydrophilic regions of the particularpolypeptide antigen. It is proposed that these regions represent thosewhich are most likely to promote T-cell or B-cell stimulation, and,hence, elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,antigen binding sites on the crystal protein-directed antibodiesdisclosed herein. Additionally or alternatively, an epitopic coresequence is one that will elicit antibodies that are cross-reactive withantibodies directed against the peptide compositions of the presentinvention. It will be understood that in the context of the presentdisclosure, the term “complementary” refers to amino acids or peptidesthat exhibit an attractive force towards each other. Thus, certainepitope core sequences of the present invention may be operationallydefined in terms of their ability to compete with or perhaps displacethe binding of the desired protein antigen with the correspondingprotein-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order of10 to 20 being more preferred. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson andWolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar® software, DNAStar, Inc., Madison, Wis.) mayalso be useful in designing synthetic peptides in accordance with thepresent disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquotted in predetermined amounts andstored in conventional manners, such as in aqueous solutions or, evenmore preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat about 4° C., or more preferably, frozen. Of course, where thepeptides are stored in a lyophilized or powdered state, they may bestored virtually indefinitely, e.g., in metered aliquots that may berehydrated with a predetermined amount of water (preferably distilled)or buffer prior to use.

2.8 Definitions

The following words and phrases have the meanings set forth below.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Pluripotent: A term used to describe develomental plasiticity. Apluripotent cell is capable of differentiating into a number ofdifferent cell types and lineages. For example, a stem cell in the bonemarrow may give rise to many different lineages of circulating bloodcells. This is in contrast to a differentiated cell, which is generallycommitted to a particular developmental pathway.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast or explant).

Structural gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence(e.g., a vector, a recombinant DNA molecule) into a cell or protoplastin which that exogenous DNA is incorporated into a chromosome or iscapable of autonomous replication.

Transformed cell: A cell whose DNA has been altered by the introductionof an exogenous DNA molecule into that cell.

Transgenic cell: Any cell derived from or regenerated from a transformedcell or derived from a transgenic cell. Exemplary transgenic cellsinclude plant calli derived from a transformed plant cell and particularcells such as leaf, root, stem, e.g., somatic cells, or reproductive(germ) cells obtained from a transgenic plant.

Transgenic plant: A plant or a progeny of any generation of the plantthat was derived from a transformed plant cell or protoplast, whereinthe plant nucleic acids contains an exogenous selected nucleic acidsequence region not originally present in a native, non-transgenic plantof the same strain. The terms “transgenic plant” and “transformed plant”have sometimes been used in the art as synonymous terms to define aplant whose DNA contains an exogenous DNA molecule. However, it isthought more scientifically correct to refer to a regenerated plant orcallus obtained from a transformed plant cell or protoplast or fromtransformed pluripotent plant cells as being a transgenic plant.Preferably, transgenic plants of the present invention include thoseplants that comprise at least a first selected polynucleotide thatencodes an insecticidal polypeptide. This selected polynucleotide ispreferably a δ-endotoxin coding region (or gene) operably linked to atleast a first promoter that expresses the coding region to produce theinsecticidal polypeptide in the transgenic plant. Preferably, thetransgenic plants of the present invention that produce the encodedpolypeptide demonstrate a phenotype of improved resistance to targetinsect pests. Such transgenic plants, their progeny, descendants, andseed from any such generation are preferably insect resistant plants.

Vector: A nucleic acid molecule capable of replication in a host celland/or to which another nucleic acid segment can be operatively linkedso as to bring about replication of the attached segment. Plasmids,phage, phagemids, and cosmids are all exemplary vectors. In manyembodiments, vectors are used as a vehicle to introduce one or moreselected polynucleotides into a host cell, thereby generating a“transformed” or “recombinant” host cell.

3.0 BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1 Restriction map of pEG1337

FIG. 2 Restriction map of pEG1921.

FIG. 3 SDS-PAGE analysis of spore-crystal suspensions from C2 culturesof EG11658, EG12156, and EG12158. Twenty-five microliters (μl) of thesuspensions were diluted with 75 μl of sterile water and prepared forelectrophoresis as described in Example 11. Ten microliters were loadedper lane on the 15% acrylamide gel. A serial dilution of bovine serumalbumin (BSA) was included as a standard. Lanes 1-3, EG11658; lanes 4-6,EG12156; lanes 7-8, EG12158. M=molecular weight standards (Sigma M-0671)in kilodaltons. The bands corresponding to CryET76, CryET80, and CryET84are indicated by the arrows.

4.0 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

4.1 Some Advantages of the Invention

The present invention provides novel δ-endotoxins which are highly toxicto insects such as WCRW, SCRW, and CPB. These protein have amino acidsequences which are only distantly related to those of otherδ-endotoxins that are toxic to dipteran or coleopteran insects. Based onthe guidelines established for the B. thuringiensis crystal proteinnomenclature (Crickmore et al., 1998), two of these polypeptides,designated CryET76 and CryET80, represent a new subclass of coleopteranactive insecticidal crystal proteins.

4.2 Insect Pests

Almost all field crops, plants, and commercial farming areas aresusceptible to attack by one or more insect pests. Particularlyproblematic are the lepidopteran and coleopteran pests identified inTable 1. For example, vegetable and cole crops such as artichokes,kohlrabi, arugula, leeks, asparagus, lentils, beans, lettuce (e.g.,head, leaf, romaine), beets, bok choy, malanga, broccoli, melons (e.g.,muskmelon, watermelon, crenshaw, honeydew, cantaloupe), brusselssprouts, cabbage, cardoni, carrots, napa, cauliflower, okra, onions,celery, parsley, chick peas, parsnips, chicory, peas, Chinese cabbage,peppers, collards, potatoes, cucumber, pumpkins, cucurbits, radishes,dry bulb onions, rutabaga, eggplant, salsify, escarole, shallots,endive, soybean, garlic, spinach, green onions, squash, greens, sugarbeets, sweet potatoes, turnip, swiss chard, horseradish, tomatoes, kale,turnips, and a variety of spices are sensitive to infestation by one ormore of the following insect pests: alfalfa looper, armyworm, beetarmyworm, artichoke plume moth, cabbage budworm, cabbage looper, cabbagewebworm, corn earworm, celery leafeater, cross-striped cabbageworm,european corn borer, diamondback moth, green cloverworm, importedcabbageworm, melonworm, omnivorous leafroller, pickleworm, rindwormcomplex, saltmarsh caterpillar, soybean looper, tobacco budworm, tomatofruitworm, tomato hornworm, tomato pinworm, velvetbean caterpillar, andyellowstriped armyworm.

Likewise, pasture and hay crops such as alfalfa, pasture grasses andsilage are often attacked by such pests as armyworm, beef armyworm,alfalfa caterpillar, European skipper, a variety of loopers andwebworms, as well as yellowstriped armyworms.

Fruit and vine crops such as apples, apricots, cherries, nectarines,peaches, pears, plums, prunes, quince almonds, chestnuts, filberts,pecans, pistachios, walnuts, citrus, blackberries, blueberries,boysenberries, cranberries, currants, loganberries, raspberries,strawberries, grapes, avocados, bananas, kiwi, persimmons, pomegranate,pineapple, tropical fruits are often susceptible to attack anddefoliation by achema sphinx moth, amorbia, armyworm, citrus cutworm,banana skipper, blackheaded fireworm, blueberry leafroller, cankerworm,cherry fruitworm, citrus cutworm, cranberry girdler, eastern tentcaterpillar, fall webworm, fall webworm, filbert leafroller, filbertwebworm, fruit tree leafroller, grape berry moth, grape leaffolder,grapeleaf skeletonizer, green fruitworm, gummosos-batrachedra commosae,gypsy moth, hickory shuckworm, hornworms, loopers, navel orangeworm,obliquebanded leafroller, omnivorous leafroller. omnivorous looper,orange tortrix, orangedog, oriental fruit moth, pandemis leafroller,peach twig borer, pecan nut casebearer, redbanded leafroller, redhumpedcaterpillar, roughskinned cutworm, saltmarsh caterpillar, spanworm, tentcaterpillar, thecla-thecla basillides, tobacco budworm, tortrix moth,tufted apple budmoth, variegated leafroller, walnut caterpillar, westerntent caterpillar, and yellowstriped armyworm.

Field crops such as canola/rape seed, evening primrose, meadow foam,corn (field, sweet, popcorn), cotton, hops, jojoba, peanuts, rice,safflower, small grains (barley, oats, rye, wheat, etc.), sorghum,soybeans, sunflowers, and tobacco are often targets for infestation byinsects including armyworm, asian and other corn borers, bandedsunflower moth, beet armyworm, bollworm, cabbage looper, corn rootworm(including southern and western varieties), cotton leaf perforator,diamondback moth, european corn borer, green cloverworm, headmoth,headworm, imported cabbageworm, loopers (including Anacamptodes spp.),obliquebanded leafroller, omnivorous leaftier, podworm, podworm,saltmarsh caterpillar, southwestern corn borer, soybean looper, spottedcutworm, sunflower moth, tobacco budworm, tobacco hornworm, velvetbeancaterpillar,

Bedding plants, flowers, ornamentals, vegetables and container stock arefrequently fed upon by a host of insect pests such as armyworm, azaleamoth, beet armyworm, diamondback moth, ello moth (hornworm), Floridafern caterpillar, Io moth, loopers, oleander moth, omnivorousleafroller, omnivorous looper, and tobacco budworm.

Forests, fruit, ornamental, and nut-bearing trees, as well as shrubs andother nursery stock are often susceptible to attack from diverse insectssuch as bagworm, blackheaded budworm, browntail moth, Californiaoakworm, douglas fir tussock moth, elm spanworm, fall webworm, fruittreeleafroller, greenstriped mapleworm, gypsy moth, jack pine budworm,mimosa webworm, pine butterfly, redhumped caterpillar, saddlebackcaterpillar, saddle prominent caterpillar, spring and fall cankerworm,spruce budworm, tent caterpillar, tortrix, and western tussock moth.Likewise, turf grasses are often attacked by pests such as armyworm, sodwebworm, and tropical sod webworm.

TABLE 1 TAXONOMY OF COLEOPTERAN PESTS IN THE SUBORDERS ARCHOSTEMATA ANDPOLYPHAGA Super- Infraorder family Family Subfamily Tribe Genus SpeciesCupedidae (reticulated Priacma P. serrata beetles) BostrichiformiaDermestidae (skin and Attagenus A. pellio larder beetles)Chrysomeliformia Cerambycidae (long- Agapanthia Agapanthia sp. hornedbeetles) Lepturinae Leptura Leptura sp. (flower long-horned beetle)Rhagium Rhagium sp. Megacyllene M. robiniae Prioninae Derobrachus D.geminatus Tetraopes T. tetropthalmus Chrysomelidae (leaf ChlamisinaeExema E. neglecta beetles) Chrysomelinae Chrysomelini Chrysomela C.tremula, Chrysomela sp. Oreina O. cacaliae Doryphorini ChrysolineChrysolina sp. Leptinotarsa L. decemlineata (Colorado potato beetle)Gonioctenini Gonioctena G. fornicata, G. holdausi, G. intermedia, G.interposita, G. kamikawai, G. linnaeana, G. nigroplagiata, G.occidentalis, G. olivacea, G. pallida, G. quinquepunctata, G.rubripennis, G. rufipes, G. tredecim-maculata, G. variabilis, G.viminalis Timarchini Timarcha Timarcha sp. Criocerinae Oulema Oulema sp.Galerucinae Galerucini Monoxia M. inornata, Monoxia sp. Ophraella O.arctica, O. artemisiae, O. bilineata, O. communa, O. conferta, O.cribrata, O. notata, O. notulata, O. nuda, O. pilosa, O. sexvittata, O.slobodkini Luperini Cerotoma C. trifurcata Diabrotica D. barberi(northern corn rootworm), D. undecimpunctata, (southern corn rootworm),D. virgifera (western corn rootworm) unclassified Lachnaia Lachnaia sp.Chrysomelidae Epitrix E. cucumeris (Harris) (potato flea beetle), E.fuscala (eggplant flea beetle) Curculionidae (weevils) CurculioninaeAnthonomus A. grandis (boll weevil) Entiminae Naupactini Aramigus A.conirostris, A. globoculus, A. intermedius, A. planioculus, A.tesselatus Otiorhynchus Otiorhynchus sp. Phyllobiini Diaprepes D.abbreviata Phyllobius Phyllobius sp. Galapaganus G. galapagoensisHyperinae Hypera H. brunneipennis (Egyptian alfalfa weevil), H. postica(alfalfa weevil), H. punctata (clover leaf weevil) Molytinae Pissodes P.affinis, P. nemorensis, P. schwarzi, P. strobi, P. terminalisRhynchophorinae Sitophilini Sitophilus S. granarius (granary weevil), S.zeamais (maize weevil) Nemonychidae Lebanorhinus L. succinus Scolytidaelps I. acuminatus, I. amitinus, I. cembrae, I. duplicatus, I.mannsfeldi, I. sexdentatus, I. typographus Orthotomicus O. erosusTomicus T. minor Cucujiformia Coccinellidae (ladybird Epilachna E.borealis (squash ladybird beetles) beetle), E. varivstis (Mexican beanbeetle) Cucujidae (flat bark Cryptolestes C. ferrugineus beetles)Oryzaephilus O. surinamensis (saw-toothed (grain grain beetle) beetles)Lagriidae (long-joined Lagria Lagria sp. beetles) Meloidae (blisterbeetles) Epicauta E. funebris Meloe M. proscarabaeus RhipiphoridaeRhipiphorus R. fasciatus Tenebrionidae (darkling Alphitobius A.diaperinus ground beetles) (lesser mealworm) Hegeter H. amaroides, H.brevicollis, H. costipennis, H. fernandezi, H. glaber, H. gomerensis, H.grancanariensis, H. impressus, H. intercedens, H. lateralis, H.plicifrons, H. politus, H. subrotundatus, H. tenuipunctatus, H.transversus, H. webbianus Misolampus M. goudoti Palorus P. ficicola, P.ratzeburgi (small- eyed flour beetle), P. subdepressus (depressed flourbeetle) Pimelia P. baetica, P. canariensis, P. criba, P. elevata, P.estevezi, P. fernan-dezlopezi, P. grandis, P. granulicollis, P. integra,P. interjecta, P. laevigata, P. lutaria, P. radula, P. sparsa, P.variolosa Tenebrio T. molitor (yellow mealworm), T. obscurus (darkmealworm) Tentyria T. schaumi Tribolium T. brevicornis, T. castaneum(red flour beetle), T. confusum (confused flour beetle), T. freemani, T.madens Zophobas Z. atratus Z. rugipes Elateriformia ElateroideaOctinodes Octinodes sp. Pyrophorus P. plagio-phthalamus ScarabaeiformiaLucanidae (Stag beetles) Dorcus D. parallelo-pipedus Lucanus L. cervusScarabaeidae Allomyrina A. dichotoma (lamellicorn beetles) CetoniinaePachnoda P. marginata (flower beetle) Dynastinae Xyloryctes X. faunusGeotrupinae Geotrupes G. stercorosus (earth-boring dung beetles)Melonlonthinae Costelytra C. zealandica (chafers) Holotrichia H.diomphalia Melolontha M. melolontha (cockchafer) Odontria O. striata O.variegata Prodontria P. bicolorata, P. capito, P. lewisi, P. tarsis, P.modesta, P. pinguis, P. praelatella, P. truncata, Prodontria sp.Scythrodes S. squalidus Rutelinae Popillia P. japonica (Japanese beetle)(shining leaf chafers) Scarabaeinae Copris C. lunaris (black dungbeetle) Scarabaeus Scarabaeus sp. (scarab) StaphyliniformiaHydrophilidae Cercyon Cercyon sp. Silphidae Nicrophorus N. americanus,N. marginatus, N. orbicollis, N. tomentosus Staphylinidae (roveCarpelimus Carpelimus sp. beetles) Quedius Q. mesomelinus TachyporusTachyporus sp. Xantholinus Xantholinus sp.4.3 Nomenclature of B. thuringiensis δ-Endotoxins

Table 2 contains a list of the traditional, and currently recognizednomenclature for the known δ-endotoxins. Also shown are GenBankaccession numbers for the sequenced polypeptides and polynucleotides.

TABLE 2 NOMENCLATURE OF KNOWN B. THURINGIENSIS δ-ENDOTOXINS^(A) New OldGenBank Accession # Cry1Aa1 CryIA(a) M11250 Cry1Aa2 CryIA(a) M10917Cry1Aa3 CryIA(a) D00348 Cry1Aa4 CryIA(a) X13535 Cry1Aa5 CryIA(a) D175182Cry1Aa6 CryIA(a) U43605 Cry1Aa7 AF081790 Cry1Aa8 I26149 Cry1Aa9 AB026261Cry1Ab1 CryIA(b) M13898 Cry1Ab2 CryIA(b) M12661 Cry1Ab3 CryIA(b) M15271Cry1Ab4 CryIA(b) D00117 Cry1Ab5 CryIA(b) X04698 Cry1Ab6 CryIA(b) M37263Cry1Ab7 CryIA(b) X13233 Cry1Ab8 CryIA(b) M16463 Cry1Ab9 CryIA(b) X54939Cry1Ab10 CryIA(b) A29125 Cry1Ab11 I12419 Cry1Ab12 AF057670 Cry1Ac1CryIA(c) M11068 Cry1Ac2 CryIA(c) M35524 Cry1Ac3 CryIA(c) X54159 Cry1Ac4CryIA(c) M73249 Cry1Ac5 CryIA(c) M73248 Cry1Ac6 CryIA(c) U43606 Cry1Ac7CryIA(c) U87793 Cry1Ac8 CryIA(c) U87397 Cry1Ac9 CryIA(c) U89872 Cry1Ac10CryIA(c) AJ002514 Cry1Ac11 AJ130970 Cry1Ac12 I12418 Cry1Ad1 CryIA(d)M73250 Cry1Ad2 A27531 Cry1Ae1 CryIA(e) M65252 Cry1Af1 U82003 Cry1Ag1AF081248 Cry1Ba1 CryIB X06711 Cry1Ba2 X95704 Cry1Bb1 ET5 L32020 Cry1Bc1CryIb(c) Z46442 Cry1Bd1 CryE1 U70726 Cry1Ca1 CryIC X07518 Cry1Ca2 CryICX13620 Cry1Ca3 CryIC M73251 Cry1Ca4 CryIC A27642 Cry1Ca5 CryIC X96682Cry1Ca6 CryIC X96683 Cry1Ca7 CryIC X96684 Cry1Cb1 CryIC(b) M97880Cry1Da1 CryID X54160 Cry1Da2 I76415 Cry1Db1 PrtB Z22511 Cry1Ea1 CryIEX53985 Cry1Ea2 CryIE X56144 Cry1Ea3 CryIE M73252 Cry1Ea4 U94323 Cry1Ea5A15535 Cry1Eb1 CryIE(b) M73253 Cry1Fa1 CryIF M63897 Cry1Fa2 CryIF M63897Cry1Fb1 PrtD Z22512 Cry1Fb2 Z22512 Cry1Fb3 AF062350 Cry1Fb4 I73895Cry1Ga1 PrtA Z22510 Cry1Ga2 CryIM Y09326 Cry1Gb1 CryH2 U70725 Cry1Ha1PrtC Z22513 Cry1Hb1 U35780 Cry1Ia1 CryV X62821 Cry1Ia2 CryV M98544Cry1Ia3 CryV L36338 Cry1Ia4 CryV L49391 Cry1Ia5 CryV Y08920 Cry1Ia6AF076953 Cry1Ib1 CryV U07642 Cry1Ic1 AF056933 Cry1Ja1 ET4 L32019 Cry1Jb1ET1 U31527 Cry1Jc1 AF056933 Cry1Ka1 U28801 Cry2Aa1 CryIIA M31738 Cry2Aa2CryIIA M23723 Cry2Aa3 D86084 Cry2Aa4 AF047038 Cry2Aa5 AJ132464 Cry2Aa6AJ1324635 Cry2Aa7 AJ132463 Cry2Ab1 CryIIB M23724 Cry2Ab2 CryIIB X55416Cry2Ac1 CryIIC X57252 Cry3Aa1 CryIIIA M22472 Cry3Aa2 CryIIIA J02978Cry3Aa3 CryIIIA Y00420 Cry3Aa4 CryIIIA M30503 Cry3Aa5 CryIIIA M37207Cry3Aa6 CryIIIA U10985 Cry3Aa7 AJ237900 Cry3Ba1 CryIIIB X17123 Cry3Ba2CryIIIB A07234 Cry3Bb1 CryIIIB2 M89794 Cry3Bb2 CryIIIC(b) U31633 Cry3Bb3I15475 Cry3Ca1 CryIIID X59797 Cry4Aa1 CryIVA Y00423 Cry4Aa2 CryIVAD00248 Cry4Ba1 CryIVB X07423 Cry4Ba2 CryIVB X07082 Cry4Ba3 CryIVB M20242Cry4Ba4 CryIVB D00247 Cry5Aa1 CryVA(a) L07025 Cry5Ab1 CryVA(b) L07026Cry5Ac1 I34543 Cry5Ba1 PS86Q3 U19725 Cry6Aa1 CryVIA L07022 Cry6Ba1CryVIB L07024 Cry7Aa1 CryIIIC M64478 Cry7Ab1 CryIIICb U04367 Cry7Ab2U04368 Cry8Aa1 CryIIIE U04364 Cry8Ba1 U04365 Cry8Ca1 U04366 Cry8Ba1CryIIIG U04365 Cry8Ca1 CryIIIF U04366 Cry9Aa1 CryIG X58120 Cry9Aa2 CryIGX58534 Cry9Ba1 CryIX X75019 Cry9Ca1 CryIH Z37527 Cry9Da1 N141 D85560Cry9Da2 AF042733 Cry9Ea1 Cry10Aa1 CryIVC M12662 Cry10Aa2 E00614 Cry11Aa1CryIVD M31737 Cry11Aa2 CryIVD M22860 Cry11Ba1 Jeg80 X86902 Cry11Bb1AF017416 Cry12Aa1 CryVB L07027 Cry13Aa1 CryVC L07023 Cry14Aa1 CryVDU13955 Cry15Aa1 34 kDa M76442 Cry16Aa1 cbm71 X94146 Cry17Aa1 cbm71X99478 Cry18Aa1 CryBP1 X99049 Cry19Aa1 Jeg65 Y08920 Cry20Aa1 U82518Cry21Aa1 I32932 Cry22Aa1 I34547 Cry23Aa1 AF03048 Cry24Aa1 U88188Cry25Aa1 U88189 Cry26Aa1 AF122897 Cry27Aa1 AB023293 Cry28Aa1 AF132928Cyt1Aa1 CytA X03182 Cyt1Aa2 CytA X04338 Cyt1Aa3 CytA Y00135 Cyt1Aa4 CytAM35968 Cyt1Ab1 CytM X98793 Cyt1Ba1 U37196 Cyt2Aa1 CytB Z14147 Cyt2Ba1“CytB” U52043 Cyt2Ba2 “CytB” AF020789 Cyt2Ba3 “CytB” AF022884 Cyt2Ba4“CytB” AF022885 Cyt2Ba5 “CytB” AF022886 Cyt2Ba6 AF034926 Cyt2Bb1 U82519Cyt2Bb1 U82519 ^(a)Adapted from: Crickmore, N. et al. Microbiol. andMol. Bio. Rev. (1998) Vol. 62: 807-813.4.4 Probes and Primers

In another aspect, DNA sequence information provided by the inventionallows for the preparation of relatively short DNA (or RNA) sequenceshaving the ability to specifically hybridize to gene sequences of theselected polynucleotides disclosed herein. In these aspects, nucleicacid probes of an appropriate length are prepared based on aconsideration of a selected crystal protein-encoding gene sequence,e.g., a sequence such as that disclosed herein. The ability of such DNAsand nucleic acid probes to specifically hybridize to a crystalprotein-encoding gene sequence lends them particular utility in avariety of embodiments. Most importantly, the probes may be used in avariety of assays for detecting the presence of complementary sequencesin a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a crystal protein gene from B. thuringiensis usingPCR™ technology. Segments of related crystal protein genes from otherspecies may also be amplified by PCR™ using such primers.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes sequences that are complementary to at leastan about 23 to about 40 or so long nucleotide stretch of a crystalprotein-encoding sequence, such as that shown herein. A size of at leastabout 14 or 15 or so nucleotides in length helps to ensure that thefragment will be of sufficient length to form a duplex molecule that isboth stable and selective. Molecules having complementary sequences overstretches greater than about 23 or so bases in length are generallypreferred, though, in order to increase stability and selectivity of thehybrid, and thereby improve the quality and degree of specific hybridmolecules obtained. One will generally prefer to design nucleic acidmolecules having gene-complementary stretches of about 14 to about 20nucleotides, or even longer where desired. Such fragments may be readilyprepared by, for example, directly synthesizing the fragment by chemicalmeans, by application of nucleic acid reproduction technology, such asthe PCR™ technology of U.S. Pat. Nos. 4,683,195, and 4,683,202,specifically incorporated herein by reference, or by excising selectedDNA fragments from recombinant plasmids containing appropriate insertsand suitable restriction sites.

4.5 Expression Vectors

The present invention contemplates a polynucleotide of the presentinvention comprised within one or more expression vectors. Thus, in oneembodiment an expression vector comprises a nucleic acid segmentcontaining a crystal protein-encoding gene operably linked to a promoterwhich expresses the gene. Additionally, the coding region may also beoperably linked to a transcription-terminating region, whereby thepromoter drives the transcription of the coding region, and thetranscription-terminating region halts transcription at some point 3′ ofthe coding region.

As used herein, the term “operatively linked” means that a promoter isconnected to an coding region in such a way that the transcription ofthat coding region is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a coding region are well known inthe art.

In a preferred embodiment, the recombinant expression of DNAs encodingthe crystal proteins of the present invention is preferable in aBacillus host cell. Preferred host cells include B. thuringiensis, B.megaterium, B. subtilis, and related bacilli, with B. thuringiensis hostcells being highly preferred. Promoters that function in bacteria arewell-known in the art. An exemplary and preferred promoter for theBacillus-derived crystal proteins include any of the known crystalprotein gene promoters, including the cry gene promoters themselves.Alternatively, mutagenized or recombinant promoters may be engineered bythe hand of man and used to promote expression of the novel genesegments disclosed herein.

In an alternate embodiment, the recombinant expression of DNAs encodingthe crystal proteins of the present invention is performed using atransformed Gram-negative bacterium such as an E. coli or Pseudomonasspp. host cell. Promoters which function in high-level expression oftarget polypeptides in E. coli and other Gram-negative host cells arealso well-known in the art.

Where an expression vector of the present invention is to be used totransform a plant, a promoter is selected that has the ability to driveexpression in plants. Promoters that function in plants are also wellknown in the art. Useful in expressing the polypeptide in plants arepromoters that are inducible, viral, synthetic, constitutive asdescribed (Poszkowski et al., 1989; Odell et al., 1985), and temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989).

A promoter is also selected for its ability to direct the transformedplant cell's or transgenic plant's transcriptional activity to thecoding region. Structural genes can be driven by a variety of promotersin plant tissues. Promoters can be near-constitutive, such as the CaMV35S promoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S,increases in polypeptide expression are found in a variety oftransformed plant tissues (e.g., callus, leaf, seed and root).Alternatively, the effects of transformation can be directed to specificplant tissues by using plant integrating vectors containing atissue-specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which isspecific for seed tissue. The Lectin protein in soybean seeds is encodedby a single gene (Le1) that is only expressed during seed maturation andaccounts for about 2 to about 5% of total seed mRNA. The lectin gene andseed-specific promoter have been fully characterized and used to directseed specific expression in transgenic tobacco plants (Vodkin et al.,1983; Lindstrom et al., 1990.)

An expression vector containing a coding region that encodes apolypeptide of interest is engineered to be under control of the lectinpromoter and that vector is introduced into plants using, for example, aprotoplast transformation method (Dhir et al., 1991a). The expression ofthe polypeptide is directed specifically to the seeds of the transgenicplant.

A transgenic plant of the present invention produced from a plant celltransformed with a tissue specific promoter can be crossed with a secondtransgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yanget al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), cornlight harvesting complex (Simpson, 1986), corn heat shock protein (Odellet al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986;Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), CaMV 35S transcript (Odell et al., 1985) andPotato patatin (Wenzler et al., 1989). Preferred promoters are thecauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunitRuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the polypeptide coding regionto which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al.,1987). However, several other plant integrating vector systems are knownto function in plants including pCaMVCN transfer control vectordescribed (Fromm et al., 1985). pCaMVCN (available from Pharmacia,Piscataway, N.J.) includes the cauliflower mosaic virus CaMV 35Spromoter.

In preferred embodiments, the vector used to express the polypeptideincludes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e., the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al., 1988).

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art.Expression (transformation vectors) used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011, the disclosures of which arespecifically incorporated herein by reference in their entirety. Thosevectors can be modified to include a coding sequence in accordance withthe present invention.

A variety of methods has been developed to operatively insert a DNAsegment into a vector via complementary cohesive termini or blunt ends.For instance, complementary homopolymer tracts can be added to the DNAsegment to be inserted and to the vector DNA. The vector and DNA segmentare then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to conferinsecticidal activity to a cell is preferably a B. thuringiensis crystalprotein-encoding gene. In preferred embodiments, such a polypeptide hasthe amino acid residue sequence of one of the sequences disclosedherein, or a functional equivalent thereof. In accordance with suchembodiments, a coding region comprising the DNA sequence of such asequence is also preferred

4.8 Nomenclature of the Novel Proteins

The inventors have arbitrarily assigned designations to the novelproteins of the invention. Likewise, the arbitrary gene designationshave been assigned to the novel nucleic acid sequence which encodesthese polypeptides. Formal assignment of gene and protein designationsbased on the revised nomenclature of crystal protein endotoxins will beassigned by a committee on the nomenclature of B. thuringiensis, formedto systematically classify B. thuringiensis crystal proteins. Theinventors contemplate that the arbitrarily assigned designations of thepresent invention will be superseded by the official nomenclatureassigned to these sequences.

4.9 Transformed Host Cells and Transgenic Plants

Methods and compositions for transforming a bacterium, a yeast cell, aplant cell, or an entire plant with one or more expression vectorscomprising a crystal protein-encoding gene segment are further aspectsof this disclosure. A transgenic bacterium, yeast cell, plant cell orplant derived from such a transformation process or the progeny andseeds from such a transgenic plant are also further embodiments of theinvention.

Means for transforming bacteria and yeast cells are well known in theart. Typically, means of transformation are similar to those well knownmeans used to transform other bacteria or yeast such as E. coli orSaccharomyces cerevisiae. Methods for DNA transformation of plant cellsinclude Agrobacterium-mediated plant transformation, protoplasttransformation, gene transfer into pollen, injection into reproductiveorgans, injection into immature embryos and particle bombardment. Eachof these methods has distinct advantages and disadvantages. Thus, oneparticular method of introducing genes into a particular plant strainmay not necessarily be the most effective for another plant strain, butit is well known which methods are useful for a particular plant strain.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973;Zatloukal et al., 1992); (2) physical methods such as microinjection(Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al.,1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston and Tang,1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al.,1993; Eglitis and Anderson, 1988a; Eglitis et al., 1988); and (4)receptor-mediated mechanisms (Curiel et al., 1991; 1992; Wagner et al.,1992).

4.9.1 Electroporation

The application of brief, high-voltage electric pulses to a variety ofanimal and plant cells leads to the formation of nanometer-sized poresin the plasma membrane. DNA is taken directly into the cell cytoplasmeither through these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of clones genes and for establishment of cell linesthat carry integrated copies of the gene of interest. Electroporation,in contrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known tothose of skill in the art. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

4.9.2 Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into maize cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered with corncells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducingdamage inflicted on the recipient cells by projectiles that are toolarge.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hr post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

4.9.3 Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNAis a relatively precise process resulting in few rearrangements. Theregion of DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., 1986; Jorgensen et al., 1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al., 1987). Therefore, commercially important cereal grains such asrice, corn, and wheat must usually be transformed using alternativemethods. However, as mentioned above, the transformation of asparagususing Agrobacterium can also be achieved (see, for example, Bytebier etal., 1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

More preferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for enhanced carboxylase activity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also bemated to produce offspring that contain two independently segregatingadded, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

4.9.4 Other Transformation Methods

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et at, 1986; Uchimiya etal., 1986; Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., 1985; Toriyama et al., 1986; Yamada etal., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1988). Inaddition, “particle gun” or high-velocity microprojectile technology canbe utilized (Vasil et al., 1992).

Using that latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metalparticles penetrate through several layers of cells and thus allow thetransformation of cells within tissue explants.

4.9.5 Gene Expression in Plants

Although great progress has been made in recent years with respect topreparation of transgenic plants which express bacterial proteins suchas B. thuringiensis crystal proteins, the results of expressing nativebacterial genes in plants are often disappointing. Unlike microbialgenetics, little was known by early plant geneticists about the factorswhich affected heterologous expression of foreign genes in plants. Inrecent years, however, several potential factors have been implicated asresponsible in varying degrees for the level of protein expression froma particular coding sequence. For example, scientists now know thatmaintaining a significant level of a particular mRNA in the cell isindeed a critical factor. Unfortunately, the causes for low steady statelevels of mRNA encoding foreign proteins are many. First, full lengthRNA synthesis may not occur at a high frequency. This could, forexample, be caused by the premature termination of RNA duringtranscription or due to unexpected mRNA processing during transcription.Second, full length RNA may be produced in the plant cell, but thenprocessed (splicing, polyA addition) in the nucleus in a fashion thatcreates a nonfunctional mRNA. If the RNA is not properly synthesized,terminated and polyadenylated, it cannot move to the cytoplasm fortranslation. Similarly, in the cytoplasm, if mRNAs have reduced halflives (which are determined by their primary or secondary sequence)insufficient protein product will be produced. In addition, there is aneffect, whose magnitude is uncertain, of translational efficiency onmRNA half-life. In addition, every RNA molecule folds into a particularstructure, or perhaps family of structures, which is determined by itssequence. The particular structure of any RNA might lead to greater orlesser stability in the cytoplasm. Structure per se is probably also adeterminant of mRNA processing in the nucleus. Unfortunately, it isimpossible to predict, and nearly impossible to determine, the structureof any RNA (except for tRNA) in vitro or in vivo. However, it is likelythat dramatically changing the sequence of an RNA will have a largeeffect on its folded structure It is likely that structure per se orparticular structural features also have a role in determining RNAstability.

To overcome these limitations in foreign gene expression, researchershave identified particular sequences and signals in RNAs that have thepotential for having a specific effect on RNA stability. In certainembodiments of the invention, therefore, there is a desire to optimizeexpression of the disclosed nucleic acid segments in planta. Oneparticular method of doing so, is by alteration of the bacterial gene toremove sequences or motifs which decrease expression in a transformedplant cell. The process of engineering a coding sequence for optimalexpression in planta is often referred to as “plantizing” a DNAsequence.

Particularly problematic sequences are those which are A+T rich.Unfortunately, since B. thuringiensis has an A+T rich genome, nativecrystal protein gene sequences must often be modified for optimalexpression in a plant. The sequence motif ATTTA (or AUUUA as it appearsin RNA) has been implicated as a destabilizing sequence in mammaliancell mRNA (Shaw and Kamen, 1986). Many short lived mRNAs have A+T rich3′ untranslated regions, and these regions often have the ATTTAsequence, sometimes present in multiple copies or as multimers (e.g.,ATTTATTTA . . . ). Shaw and Kamen showed that the transfer of the 3′ endof an unstable mRNA to a stable RNA (globin or VA1) decreased the stableRNA's half life dramatically. They further showed that a pentamer ofATTTA had a profound destabilizing effect on a stable message, and thatthis signal could exert its effect whether it was located at the 3′ endor within the coding sequence. However, the number of ATTTA sequencesand/or the sequence context in which they occur also appear to beimportant in determining whether they function as destabilizingsequences. Shaw and Kamen showed that a trimer of ATTTA had much lesseffect than a pentamer on mRNA stability and a dimer or a monomer had noeffect on stability (Shaw and Kamen, 1987). Note that multimers of ATTTAsuch as a pentamer automatically create an A+T rich region. This wasshown to be a cytoplasmic effect, not nuclear. In other unstable mRNAs,the ATTTA sequence may be present in only a single copy, but it is oftencontained in an A+T rich region. From the animal cell data collected todate, it appears that ATTTA at least in some contexts is important instability, but it is not yet possible to predict which occurrences ofATTTA are destabiling elements or whether any of these effects arelikely to be seen in plants.

Some studies on mRNA degradation in animal cells also indicate that RNAdegradation may begin in some cases with nucleolytic attack in A+T richregions. It is not clear if these cleavages occur at ATTTA sequences.There are also examples of mRNAs that have differential stabilitydepending on the cell type in which they are expressed or on the stagewithin the cell cycle at which they are expressed. For example, histonemRNAs are stable during DNA synthesis but unstable if DNA synthesis isdisrupted. The 3′ end of some histone mRNAs seems to be responsible forthis effect (Pandey and Marzluff, 1987). It does not appear to bemediated by ATTTA, nor is it clear what controls the differentialstability of this mRNA. Another example is the differential stability ofIgG mRNA in B lymphocytes during B cell maturation (Genovese andMilcarek, 1988). These examples all provide evidence that mRNA stabilitycan be mediated by cell type or cell cycle specific factors. Furthermorethis type of instability is not yet associated with specific sequences.Given these uncertainties, it is not possible to predict which RNAs arelikely to be unstable in a given cell. In addition, even the ATTTA motifmay act differentially depending on the nature of the cell in which theRNA is present. Shaw and Kamen (1987) have reported that activation ofprotein kinase C can block degradation mediated by ATTTA.

The addition of a polyadenylate string to the 3′ end is common to mosteukaryotic mRNAs, both plant and animal. The currently accepted view ofpolyA addition is that the nascent transcript extends beyond the mature3′ terminus. Contained within this transcript are signals forpolyadenylation and proper 3′ end formation. This processing at the 3′end involves cleavage of the mRNA and addition of polyA to the mature 3′end. By searching for consensus sequences near the polyA tract in bothplant and animal mRNAs, it has been possible to identify consensussequences that apparently are involved in polyA addition and 3′ endcleavage. The same consensus sequences seem to be important to both ofthese processes. These signals are typically a variation on the sequenceAATAAA. In animal cells, some variants of this sequence that arefunctional have been identified; in plant cells there seems to be anextended range of functional sequences (Wickens and Stephenson, 1984;Dean et al., 1986). Because all of these consensus sequences arevariations on AATAAA, they all are A+T rich sequences. This sequence istypically found 15 to 20 bp before the polyA tract in a mature mRNA.Studies in animal cells indicate that this sequence is involved in bothpolyA addition and 3′ maturation. Site directed mutations in thissequence can disrupt these functions (Conway and Wickens, 1988; Wickenset al., 1987). However, it has also been observed that sequences up to50 to 100 bp 3′ to the putative polyA signal are also required; i.e. agene that has a normal AATAAA but has been replaced or disrupteddownstream does not get properly polyadenylated (Gil and Proudfoot,1984; Sadofsky and Alwine, 1984; McDevitt et al., 1984). That is, thepolyA signal itself is not sufficient for complete and properprocessing. It is not yet known what specific downstream sequences arerequired in addition to the polyA signal, or if there is a specificsequence that has this function. Therefore, sequence analysis can onlyidentify potential polyA signals.

In naturally occurring mRNAs that are normally polyadenylated, it hasbeen observed that disruption of this process, either by altering thepolyA signal or other sequences in the mRNA, profound effects can beobtained in the level of functional mRNA. This has been observed inseveral naturally occurring mRNAs, with results that are gene-specificso far.

It has been shown that in natural mRNAs proper polyadenylation isimportant in mRNA accumulation, and that disruption of this process caneffect mRNA levels significantly. However, insufficient knowledge existsto predict the effect of changes in a normal gene. In a heterologousgene, it is even harder to predict the consequences. However, it ispossible that the putative sites identified are dysfunctional. That is,these sites may not act as proper polyA sites, but instead function asaberrant sites that give rise to unstable mRNAs.

In animal cell systems, AATAAA is by far the most common signalidentified in mRNAs upstream of the polyA, but at least four variantshave also been found (Wickens and Stephenson, 1984). In plants, notnearly so much analysis has been done, but it is clear that multiplesequences similar to AATAAA can be used. The plant sites in Table 3called major or minor refer only to the study of Dean et al. (1986)which analyzed only three types of plant gene. The designation ofpolyadenylation sites as major or minor refers only to the frequency oftheir occurrence as functional sites in naturally occurring genes thathave been analyzed. In the case of plants this is a very limiteddatabase. It is hard to predict with any certainty that a sitedesignated major or minor is more or less likely to function partiallyor completely when found in a heterologous gene such as those encodingthe crystal proteins of the present invention.

TABLE 3 POLYADENYLATION SITES IN PLANT GENES PA AATAAA Major consensussite P1A AATAAT Major plant site P2A AACCAA Minor plant site P3A ATATAAMinor plant site P4A AATCAA Minor plant site P5A ATACTA Minor plant siteP6A ATAAAA Minor plant site P7A ATGAAA Minor plant site P8A AAGCAT Minorplant site P9A ATTAAT Minor plant site P10A ATACAT Minor plant site P11AAAAATA Minor plant site P12A ATTAAA Minor animal site P13A AATTAA Minoranimal site P14A AATACA Minor animal site P15A CATAAA Minor animal site

The present invention provides a method for preparing synthetic plantgenes which genes express their protein product at levels significantlyhigher than the wild-type genes which were commonly employed in planttransformation heretofore. In another aspect, the present invention alsoprovides novel synthetic plant genes which encode non-plant proteins.

As described above, the expression of native B. thuringiensis genes inplants is often problematic. The nature of the coding sequences of B.thuringiensis genes distinguishes them from plant genes as well as manyother heterologous genes expressed in plants. In particular, B.thuringiensis genes are very rich (˜62%) in adenine (A) and thymine (T)while plant genes and most other bacterial genes which have beenexpressed in plants are on the order of 45-55% A+T.

Due to the degeneracy of the genetic code and the limited number ofcodon choices for any amino acid, most of the “excess” A+T of thestructural coding sequences of some Bacillus species are found in thethird position of the codons. That is, genes of some Bacillus specieshave A or T as the third nucleotide in many codons. Thus A+T content inpart can determine codon usage bias. In addition, it is clear that genesevolve for maximum function in the organism in which they evolve. Thismeans that particular nucleotide sequences found in a gene from oneorganism, where they may play no role except to code for a particularstretch of amino acids, have the potential to be recognized as genecontrol elements in another organism (such as transcriptional promotersor terminators, polyA addition sites, intron splice sites, or specificmRNA degradation signals). It is perhaps surprising that such misreadsignals are not a more common feature of heterologous gene expression,but this can be explained in part by the relatively homogeneous A+Tcontent (˜50%) of many organisms. This A+T content plus the nature ofthe genetic code put clear constraints on the likelihood of occurrenceof any particular oligonucleotide sequence. Thus, a gene from E. coliwith a 50% A+T content is much less likely to contain any particular A+Trich segment than a gene from B. thuringiensis.

Typically, to obtain high-level expression of the δ-endotoxin genes inplants, existing structural coding sequence (“structural gene”) whichcodes for the δ-endotoxin are modified by removal of ATTTA sequences andputative polyadenylation signals by site directed mutagenesis of the DNAcomprising the structural gene. It is most preferred that substantiallyall the polyadenylation signals and ATTTA sequences are removed althoughenhanced expression levels are observed with only partial removal ofeither of the above identified sequences. Alternately if a syntheticgene is prepared which codes for the expression of the subject protein,codons are selected to avoid the ATTTA sequence and putativepolyadenylation signals. For purposes of the present invention putativepolyadenylation signals include, but are not necessarily limited to,AATAAA, AATAAT, AACCAA, ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT,ATTAAT, ATACAT, AAAATA, ATTAAA, AATTAA, AATACA and CATAAA. In replacingthe ATTTA sequences and polyadenylation signals, codons are preferablyutilized which avoid the codons which are rarely found in plant genomes.

The selected DNA sequence is scanned to identify regions with greaterthan four consecutive adenine (A) or thymine (T) nucleotides. The A+Tregions are scanned for potential plant polyadenylation signals.Although the absence of five or more consecutive A or T nucleotideseliminates most plant polyadenylation signals, if there are more thanone of the minor polyadenylation signals identified within tennucleotides of each other, then the nucleotide sequence of this regionis preferably altered to remove these signals while maintaining theoriginal encoded amino acid sequence.

The second step is to consider the about 15 to about 30 or so nucleotideresidues surrounding the A+T rich region identified in step one. If theA+T content of the surrounding region is less than 80%, the regionshould be examined for polyadenylation signals. Alteration of the regionbased on polyadenylation signals is dependent upon (1) the number ofpolyadenylation signals present and (2) presence of a major plantpolyadenylation signal.

The extended region is examined for the presence of plantpolyadenylation signals. The polyadenylation signals are removed bysite-directed mutagenesis of the DNA sequence. The extended region isalso examined for multiple copies of the ATTTA sequence which are alsoremoved by mutagenesis.

It is also preferred that regions comprising many consecutive A+T basesor G+C bases are disrupted since these regions are predicted to have ahigher likelihood to form hairpin structure due to self-complementarity.Therefore, insertion of heterogeneous base pairs would reduce thelikelihood of self-complementary secondary structure formation which areknown to inhibit transcription and/or translation in some organisms. Inmost cases, the adverse effects may be minimized by using sequenceswhich do not contain more than five consecutive A+T or G+C.

4.9.6 Synthetic Oligonucleotides for Mutagenesis

When oligonucleotides are used in the mutagenesis, it is desirable tomaintain the proper amino acid sequence and reading frame, withoutintroducing common restriction sites such as BglII, HindIII, SacI, KpnI,EcoRI, NcoI, PstI and SalI into the modified gene. These restrictionsites are found in poly-linker insertion sites of many cloning vectors.Of course, the introduction of new polyadenylation signals, ATTTAsequences or consecutive stretches of more than five A+T or G+C, shouldalso be avoided. The preferred size for the oligonucleotides is about 40to about 50 bases, but fragments ranging from about 18 to about 100bases have been utilized. In most cases, a minimum of about 5 to about 8base pairs of homology to the template DNA on both ends of thesynthesized fragment are maintained to insure proper hybridization ofthe primer to the template. The oligonucleotides should avoid sequenceslonger than five base pairs A+T or G+C. Codons used in the replacementof wild-type codons should preferably avoid the TA or CG doubletwherever possible. Codons are selected from a plant preferred codontable (such as Table 4 below) so as to avoid codons which are rarelyfound in plant genomes, and efforts should be made to select codons topreferably adjust the G+C content to about 50%.

TABLE 4 PREFERRED CODON USAGE IN PLANTS Amino Percent Usage Acid Codonin Plants ARG CGA 7 CGC 11 CGG 5 CGU 25 AGA 29 AGG 23 LEU CUA 8 CUC 20CUG 10 CUU 28 UUA 5 UUG 30 SER UCA 14 UCC 26 UCG 3 UCU 21 AGC 21 AGU 15THR ACA 21 ACC 41 ACG 7 ACU 31 PRO CCA 45 CCC 19 CCG 9 CCU 26 ALA GCA 23GCC 32 GCG 3 GCU 41 GLY GGA 32 GGC 20 GGG 11 GGU 37 ILE AUA 12 AUC 45AUU 43 VAL GUA 9 GUC 20 GUG 28 GUU 43 LYS AAA 36 AAG 64 ASN AAC 72 AAU28 GLN CAA 64 CAG 36 HIS CAC 65 CAU 35 GLU GAA 48 GAG 52 ASP GAC 48 GAU52 TYR UAC 68 UAU 32 CYS UGC 78 UGU 22 PHE UUC 56 UUU 44 MET AUG 100 TRPUGG 100

Regions with many consecutive A+T bases or G+C bases are predicted tohave a higher likelihood to form hairpin structures due toself-complementarity. Disruption of these regions by the insertion ofheterogeneous base pairs is preferred and should reduce the likelihoodof the formation of self-complementary secondary structures such ashairpins which are known in some organisms to inhibit transcription(transcriptional terminators) and translation (attenuators).

Alternatively, a completely synthetic gene for a given amino acidsequence can be prepared, with regions of five or more consecutive A+Tor G+C nucleotides being avoided. Codons are selected avoiding the TAand CG doublets in codons whenever possible. Codon usage can benormalized against a plant preferred codon usage table (such as Table 4)and the G+C content preferably adjusted to about 50%. The resultingsequence should be examined to ensure that there are minimal putativeplant polyadenylation signals and ATTTA sequences. Restriction sitesfound in commonly used cloning vectors are also preferably avoided.However, placement of several unique restriction sites throughout thegene is useful for analysis of gene expression or construction of genevariants.

4.10 Methods for Producing Insect-Resistant Transgenic Plants

By transforming a suitable host cell, such as a plant cell, with arecombinant cry gene-containing segment, the expression of the encodedcrystal protein (i.e. a bacterial crystal protein or polypeptide havinginsecticidal activity against Coleopterans) can result in the formationof insect-resistant plants.

By way of example, one may utilize an expression vector containing acoding region for a B. thuringiensis crystal protein and an appropriateselectable marker to transform a suspension of embryonic plant cells,such as wheat or corn cells using a method such as particle bombardment(Maddock et al., 1991; Vasil et al., 1992) to deliver the DNA coated onmicroprojectiles into the recipient cells. Transgenic plants are thenregenerated from transformed embryonic calli that express theinsecticidal proteins.

The formation of transgenic plants may also be accomplished using othermethods of cell transformation which are known in the art such asAgrobacterium-mediated DNA transfer (Fraley et al., 1983).Alternatively, DNA can be introduced into plants by direct DNA transferinto pollen (Zhou et al., 1983; Hess, 1987; Luo et al., 1988), byinjection of the DNA into reproductive organs of a plant (Pena et al.,1987), or by direct injection of DNA into the cells of immature embryosfollowed by the rehydration of desiccated embryos (Neuhaus et al., 1987;Benbrook et al., 1986).

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, 1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983).

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

A transgenic plant of this invention thus has an increased amount of acoding region (e.g., a gene) that encodes a polypeptide as disclosedherein. A preferred transgenic plant is an independent segregant and cantransmit that gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for that gene, and transmits that gene toall of its offspring on sexual mating. Seed from a transgenic plant maybe grown in the field or greenhouse, and resulting sexually maturetransgenic plants are self-pollinated to generate true breeding plants.The progeny from these plants become true breeding lines that areevaluated for, by way of example, increased insecticidal capacityagainst coleopteran insects, preferably in the field, under a range ofenvironmental conditions. The inventors contemplate that the presentinvention will find particular utility in the creation of transgenicplants of commercial interest including various turf and pasturegrasses, rye, wheat, corn, kapok, flax, rice, barley, oats, sugarcane,cotton, tomato, potato, soybeans and other legumes, tobacco, sorghum, aswell as a variety of ornamental plants including cacti and succulents,fruits, berries, vegetables, and also a number of nut- and fruit-bearingtrees and plants.

Transgenic plants comprising one or more transgenes that encode apolypeptide as described herein will preferably exhibit a phenotype ofimproved or enhanced insect resistance to the target coleopteran andlepidopteran insects as described herein. These plants will preferablyprovide transgenic seeds, which will be used to create lineages oftransgenic plants (i.e. progeny or advanced generations of the originaltransgenic plant) that may be used to produce seed, or used as animal orhuman foodstuffs, or to produce fibers, oil, fruit, grains, or othercommercially-important plant products or plant-derived components. Insuch instances, the progeny and seed obtained from any generation of thetransformed plants will contain the selected chromosomally-integratedtransgene that encodes the δ-endotoxin of the present invention. Thetransgenic plants of the present invention may be crossed to producehybrid or inbred lines with one or more plants that have desirableproperties. In certain circumstances, it may also be desirable to createtransgenic plants, seed, and progeny that contain one or more additionaltransgenes incorporated into their genome in addition to the transgeneencoding the polypeptide of the invention. For example, the transgenicplants may contain a second gene encoding the same, or a differentinsect-resistance polypeptide, or alternatively, the plants may compriseone or more additional transgenes such as those conferring herbicideresistance, fungal resistance, bacterial resistance, stress, salt, ordrought tolerance, improved stalk or root lodging, increased starch,grain, oil, carbohydrate, amino acid, protein production, and the like.

4.11 Isolating Homologous Gene and Gene Fragments

The genes and δ-endotoxins according to the subject invention includenot only the full length sequences disclosed herein but also fragmentsof these sequences, or fusion proteins, which retain the characteristicinsecticidal activity of the sequences specifically exemplified herein.

It should be apparent to a person skill in this art that insecticidalδ-endotoxins can be identified and obtained through several means. Thespecific genes, or portions thereof, may be obtained from a culturedepository, or constructed synthetically, for example, by use of a genemachine. Variations of these genes may be readily constructed usingstandard techniques for making point mutations. Also, fragments of thesegenes can be made using commercially available exonucleases orendonucleases according to standard procedures. For example, enzymessuch as Bal31 or site-directed mutagenesis can be used to systematicallycut off nucleotides from the ends of these genes. Also, genes which codefor active fragments may be obtained using a variety of otherrestriction enzymes. Proteases may be used to directly obtain activefragments of these δ-endotoxins.

Equivalent δ-endotoxins and/or genes encoding these equivalentδ-endotoxins can also be isolated from Bacillus strains and/or DNAlibraries using the teachings provided herein. For example, antibodiesto the δ-endotoxins disclosed and claimed herein can be used to identifyand isolate other δ-endotoxins from a mixture of proteins. Specifically,antibodies may be raised to the portions of the δ-endotoxins which aremost constant and most distinct from other B. thuringiensisδ-endotoxins. These antibodies can then be used to specifically identifyequivalent δ-endotoxins with the characteristic insecticidal activity byimmunoprecipitation, enzyme linked immunoassay (ELISA), or Westernblotting.

A further method for identifying the δ-endotoxins and genes of thesubject invention is through the use of oligonucleotide probes. Theseprobes are nucleotide sequences having a detectable label. As is wellknown in the art, if the probe molecule and nucleic acid samplehybridize by forming a strong bond between the two molecules, it can bereasonably assumed that the probe and sample are essentially identical.The probe's detectable label provides a means for determining in a knownmanner whether hybridization has occurred. Such a probe analysisprovides a rapid method for identifying formicidal δ-endotoxin genes ofthe subject invention.

The nucleotide segments which are used as probes according to theinvention can be synthesized by use of DNA synthesizers using standardprocedures. In the use of the nucleotide segments as probes, theparticular probe is labeled with any suitable label known to thoseskilled in the art, including radioactive and non-radioactive labels.Typical radioactive labels include ³²P, ¹²⁵I, ³⁵S, or the like. A probelabeled with a radioactive isotope can be constructed from a nucleotidesequence complementary to the DNA sample by a conventional nicktranslation reaction, using a DNase and DNA polymerase. The probe andsample can then be combined in a hybridization buffer solution and heldat an appropriate temperature until annealing occurs. Thereafter, themembrane is washed free of extraneous materials, leaving the sample andbound probe molecules typically detected and quantified byautoradiography and/or liquid scintillation counting.

Non-radioactive labels include, for example, ligands such as biotin orthyroxin, as well as enzymes such as hydrolases or peroxidases, or thevarious chemiluminescers such as luciferin, or fluorescent compoundslike fluorescein and its derivatives. The probe may also be labeled atboth ends with different types of labels for ease of separation, as, forexample, by using an isotopic label at the end mentioned above and abiotin label at the other end.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probes of thesubject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, by methods currently known to anordinarily skilled artisan, and perhaps by other methods which maybecome known in the future.

The potential variations in the probes listed is due, in part, to theredundancy of the genetic code. Because of the redundancy of the geneticcode, i.e. more than one coding nucleotide triplet (codon) can be usedfor most of the amino acids used to make proteins. Therefore differentnucleotide sequences can code for a particular amino acid. Thus, theamino acid sequences of the B. thuringiensis δ-endotoxins and peptidescan be prepared by equivalent nucleotide sequences encoding the sameamino acid sequence of the protein or peptide. Accordingly, the subjectinvention includes such equivalent nucleotide sequences. Also, inverseor complement sequences are an aspect of the subject invention and canbe readily used by a person skilled in this art. In addition it has beenshown that proteins of identified structure and function may beconstructed by changing the amino acid sequence if such changes do notalter the protein secondary structure (Kaiser and Kezdy, 1984). Thus,the subject invention includes mutants of the amino acid sequencedepicted herein which do not alter the protein secondary structure, orif the structure is altered, the biological activity is substantiallyretained. Further, the invention also includes mutants of organismshosting all or part of a δ-endotoxin encoding a gene of the invention.Such mutants can be made by techniques well known to persons skilled inthe art. For example, UV irradiation can be used to prepare mutants ofhost organisms. Likewise, such mutants may include asporogenous hostcells which also can be prepared by procedures well known in the art.

4.13 Recombinant Host Cells

The nucleotide sequences of the subject invention may be introduced intoa wide variety of microbial and eukaryotic hosts. As hosts forrecombinant expression of Cry polypeptides, of particular interest willbe the prokaryotes and the lower eukaryotes, such as fungi. Illustrativeprokaryotes, both Gram-negative and Gram-positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae, Actinomycetales, andNitrobacteraceae. Among eukaryotes are fungi, such as Phycomycetes andAscomycetes, which includes yeast, such as Saccharomyces andSchizosaccharomyces; and Basidiomycetes yeast, such as Rhodotorula,Aureobasidium, Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the geneticconstructs of the present invention into the host cell, availability ofexpression systems, efficiency of expression, stability of the gene ofinterest in the host, and the presence of auxiliary geneticcapabilities.

A large number of microorganisms known to inhabit the phylloplane (thesurface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops may alsobe desirable host cells for manipulation, propagation, storage, deliveryand/or mutagenesis of the disclosed genetic constructs. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Bacillus(including the species and subspecies B. thuringiensis kurstaki HD-1, B.thuringiensis kurstaki HD-73, B. thuringiensis sotto, B. thuringiensisberliner, B. thuringiensis thuringiensis, B. thuringiensis tolworthi, B.thuringiensis dendrolimus, B. thuringiensis alesti, B. thuringiensisgalleriae, B. thuringiensis aizawai, B. thuringiensis subtoxicus, B.thuringiensis entomocidus, B. thuringiensis tenebrionis and B.thuringiensis san diego); Pseudomonas, Erwinia, Serratia, Klebsiella,Zanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius,Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter,Leuconostoc, and Alcaligenes; fungi, particularly yeast, e.g., generaSaccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula,and Aureobasidium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodobactersphaeroides, Xanthomonas campestris, Rhizobium melioti, Alcaligeneseutrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing a selected geneticconstruct into the host, availability of expression systems, efficiencyof expression, stability of the polynucleotide in the host, and thepresence of auxiliary genetic capabilities. Other considerations includeease of formulation and handling, economics, storage stability, and thelike.

4.14 Polynucleotide Sequences

DNA compositions encoding the insecticidally-active polypeptides of thepresent invention are particularly preferred for delivery to recipientplant cells, in the generation of pluripotent plant cells, andultimately in the production of insect-resistant transgenic plants. Forexample, DNA segments in the form of vectors and plasmids, or linear DNAfragments, in some instances containing only the DNA element to beexpressed in the plant cell, and the like, may be employed.

Vectors, plasmids, phagemids, cosmids, viral vectors, shuttle vectors,baculovirus vectors, BACs (bacterial artificial chromosomes), YACs(yeast artificial chromosomes) and DNA segments for use in transformingcells with a δ-endotoxin-encoding polynucleotide, will, of course,generally comprise at least a first gene that encodes the polypeptide inaccordance with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:19, ora gene that encodes a polypeptide that has at least about 80% or 85% or90% or 95% sequence identity to the amino acid sequence disclosed in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:19. These nucleic acidconstructs may comprise one or more genes which one desires to introduceinto recipient cells. These DNA constructs can include structures suchas promoters, enhancers, polylinkers, or regulatory genes as desired.The DNA segment or gene chosen for cellular introduction will oftenencode a polypeptide which will be expressed in the resultantrecombinant cells, such as will result in a screenable or selectabletrait and/or which will impart an improved phenotype to the transformedhost cell. Alternatively, the nucleic acid constructs may containantisense constructs, or ribozyme-encoding regions when delivery orintroduction of such nucleic acid constructs are desirable.

4.15 Methods for Preparing Mutagenized Polynucleotides

In certain circumstances, it may be desirable to modify or alter one ormore nucleotides in one or more of the polynucleotide sequencesdisclosed herein for the purpose of altering or changing theinsecticidal activity or insecticidal specificity of the encodedpolypeptide. The mutant sequence is then subsequently amplified. Methodsfor mutagenizing and amplifying a DNA segment are well-known to those ofskill in the art. Mutagenesis of the DNA segments may be made by randomor site-specific mutagenesis procedures. The polynucleotides may bemodified by the addition, deletion, or substitution of one or morenucleotides from the sequence encoding the insecticidally-activepolypeptide.

Particular mutagenesis and amplification methods which may be useful inthe practice of the present invention are described by Tomic et al.,Michael, et al., Upender et al., Kwoh et al., Frohman, et al., Ohara etal., Wu, et al., Walker et al.; U.S. Pat. Nos. 4,683,195, 4,683,202,4,800,159, 4,883,750, and; EP320,308 EP329,822; GB 2202328;PCT/US87/00880; PCT/US89/01025; WO 88/10315, WO 89/06700, each of whichis incorporated herein by reference in its entirety.

4.16 Post-Transcriptional Events Affecting Expression of Transgenes inPlants

In many instances, the level of transcription of a particular transgenein a given host cell is not always indicative of the amount of proteinbeing produced in the transformed host cell. This is often due topost-transcriptional processes, such as splicing, polyadenylation,appropriate translation initiation, and RNA stability, that affect theability of a transcript to produce protein. Such factors may also affectthe stability and amount of mRNA produced from the given transgene. Assuch, it is often desirable to alter the post-translational eventsthrough particular molecular biology techniques. The inventorscontemplate that in certain instances it may be desirable to alter thetranscription and/or expression of the polypeptide-encoding nucleic acidconstructs of the present invention to increase, decrease, or otherwiseregulate or control these constructs in particular host cells and/ortransgenic plants.

4.16.1 Efficient Initiation of Protein Translation

The 5′-untranslated leader (5′-UTL) sequence of eukaryotic mRNA plays amajor role in translational efficiency. Many early chimeric transgenesusing a viral promoter used an arbitrary length of viral sequence afterthe transcription initiation site and fused this to the AUG of thecoding region. More recently studies have shown that the 5′-UTL sequenceand the sequences directly surrounding the AUG can have a large effectin translational efficiency in host cells and particularly certain plantspecies and that this effect can be different depending on theparticular cells or tissues in which the message is expressed.

In most eukaryotic mRNAs, the point of translational initiation occursat the AUG codon closest to the 5′ cap of the transcript. Comparison ofplant mRNA sequences and site directed mutagenesis experiments havedemonstrated the existence of a consensus sequence surrounding theinitiation codon in plants, 5′-UAAACAAUGGCU-3′ (SEQ ID NO:35) (Joshi,1987; Lutcke et al., 1987). However, consensus sequences will beapparent amongst individual plant species. For example, a compilation ofsequences surrounding the initiation codon from 85 maize genes yields aconsensus of 5′-(C/G)AUGGCG-3′ (Luehrsen et al., 1994). In tobaccoprotoplasts, transgenes encoding β-glucuronidase (GUS) and bacterialchitinase showed a 4-fold and an 8-fold increase in expression,respectively, when the native sequences of these genes were changed toencode 5′-ACCAUGG-3′ (Gallie et al., 1987b; Jones et al., 1988).

When producing chimeric transgenes (i.e. transgenes comprising DNAsegments from different sources operably linked together), often the5′-UTL of plant viruses are used. The alfalfa mosaic virus (AMV) coatprotein and brome mosaic virus (BMV) coat protein 5′-UTLs have beenshown to enhance mRNA translation 8-fold in electroporated tobaccoprotoplasts (Gallie et al., 1987a; 1987b). A 67-nucleotide derivative(Ω) of the 5′-UTL of tobacco mosaic virus RNA (TMV) fused to thechloramphenicol acetyltransferase (CAT) gene and GUS gene has been shownto enhance translation of reporter genes in vitro (Gallie et al., 1987a;1987b; Sleat et al., 1987; Sleat et al., 1988). Electroporation oftobacco mesophyll protoplasts with transcripts containing the TMV leaderfused to reporter genes CAT, GUS, and LUC produced a 33-, 21-, and36-fold level of enhancement, respectively (Gallie et al., 1987a; 1987b;Gallie et al., 1991). Also in tobacco, an 83-nt 5′-UTL of potato virus XRNA was shown to enhance expression of the neomycin phosphotransfereseII (NptII) 4-fold (Poogin and Skryabin, 1992).

The effect of a 5′-UTL may be different depending on the plant,particularly between dicots and monocots. The TMV 5′-UTL has been shownto be more effective in tobacco protoplasts (Gallie et al., 1989) thanin maize protoplasts (Gallie and Young, 1994). Also, the 5′-UTLs fromTMV-Ω (Gallie et al., 1988), AMV-coat (Gehrke et al., 1983; Jobling andGehrke, 1987), TMV-coat (Goelet et al., 1982), and BMV-coat (French etal., 1986) worked poorly in maize and inhibited expression of aluciferase gene in maize relative to its native leader (Koziel et al.,1996). However, the 5′-UTLs from the cauliflower mosaic virus (CaMV) 35Stranscript and the maize genes glutelin (Boronat et al., 1986),PEP-carboxylase (Hudspeth and Grula, 1989) and ribulose biphosphatecarboxylase showed a considerable increase in expression of theluciferase gene in maize relative to its native leader (Koziel et al.,1996).

These 5′-UTLs had different effects in tobacco. In contrast to maize,the TMV Ω 5′-UTL and the AMV coat protein 5′-UTL enhanced expression intobacco, whereas the glutelin, maize PEP-carboxylase and maizeribulose-1,5-bisphosphate carboxylase 5′-UTLs did not show enhancementrelative to the native luciferase 5′-UTL (Koziel et al., 1996). Only theCaMV 35S 5′-UTL enhanced luciferase expression in both maize and tobacco(Koziel et al., 1996). Furthermore, the TMV and BMV coat protein 5′-UTLswere inhibitory in both maize and tobacco protoplasts (Koziel et al.,1996).

4.16.2 Use of Introns to Increase Expression

Including one or more introns in the transcribed portion of a gene hasbeen found to increase heterologous gene expression in a variety ofplant systems (Callis et al., 1987; Maas et al., 1991; Mascerenhas etal., 1990; McElroy et al., 1990; Vasil et al., 1989), although not allintrons produce a stimulatory effect and the degree of stimulationvaries. The enhancing effect of introns appears to be more apparent inmonocots than in dicots. Tanaka et al., (1990) has shown that use of thecatalase intron 1 isolated from castor beans increases gene expressionin rice. Likewise, the first intron of the alcohol dehydrogenase 1(Adh1) has been shown to increase expression of a genomic clone of Adh1comprising the endogenous promoter in transformed maize cells (Callis etal., 1987; Dennis et al., 1984). Other introns that are also able toincrease expression of transgenes which contain them include the introns2 and 6 of Adh1 (Luehrsen and Walbot, 1991), the catalase intron (Tanakaet al., 1990), intron 1 of the maize bronze 1 gene (Callis et al.,1987), the maize sucrose synthase intron 1 (Vasil et al., 1989), intron3 of the rice actin gene (Luehrsen and Walbot, 1991), rice actin intron1 (McElroy et al., 1990), and the maize ubiquitin exon 1 (Christensen etal., 1992).

Generally, to achieve optimal expression, the selected intron(s) shouldbe present in the 5′ transcriptional unit in the correct orientationwith respect to the splice junction sequences (Callis et al., 1987; Maaset al., 1991; Mascerenhas et al., 1990; Oard et al., 1989; Tanaka etal., 1990; Vasil et al., 1989). Intron 9 of Adh1 has been shown toincrease expression of a heterologous gene when placed 3′ (or downstreamof) the gene of interest (Callis et al., 1987).

4.16.3 Use of Synthetic Genes to Increase Expression of HeterologousGenes in Plants

When introducing a prokaryotic gene into a eukaryotic host, or whenexpressing a eukaryotic gene in a non-native host, the sequence of thegene must often be altered or modified to allow efficient translation ofthe transcript(s) derived form the gene. Significant experience in usingsynthetic genes to increase expression of a desired protein has beenachieved in the expression of B. thuringiensis in plants. Native B.thuringiensis genes are often expressed only at low levels in dicots andsometimes not at all in many species of monocots (Koziel et al., 1996).Codon usage in the native genes is considerably different from thatfound in typical plant genes, which have a higher G+C content.Strategies to increase expression of these genes in plants generallyalter the overall G+C content of the genes. For example, synthetic B.thuringiensis crystal-protein encoding genes have resulted insignificant improvements in expression of these endotoxins in variouscrops including cotton (Perlak et al., 1990; Wilson et al., 1992),tomato (Perlak et al., 1991), potato (Perlak et al., 1993), rice (Chenget al., 1998), and maize (Koziel et al., 1993).

In a similar fashion the inventors contemplate that the geneticconstructs of the present invention, because they contain one or moregenes of bacterial origin, may in certain circumstances be altered toincrease the expression of these prokaryotic-derived genes in particulareukaryotic host cells and/or transgenic plants which comprise suchconstructs. Using molecular biology techniques which are well-known tothose of skill in the art, one may alter the coding or non codingsequences of the particular Cry-encoding gene sequences to optimize orfacilitate its expression in transformed plant cells at levels suitablefor preventing or reducing insect infestation or attack in suchtransgenic plants.

4.16.4 Chloroplast Sequestering and Targeting

Another approach for increasing expression of A+T rich genes in plantshas been demonstrated in tobacco chloroplast transformation. High levelsof expression of an unmodified B. thuringiensis crystal protein-encodinggenes in tobacco has been reported by McBride et al., (1995).

Additionally, methods of targeting proteins to the chloroplast have beendeveloped. This technique, utilizing the pea chloroplast transitpeptide, has been used to target the enzymes of the polyhydroxybutyratesynthesis pathway to the chloroplast (Nawrath et al., 1994). Also, thistechnique negated the necessity of modification of the coding regionother than to add an appropriate targeting sequence.

U.S. Pat. No. 5,576,198 (specifically incorporated herein by reference)discloses compositions and methods useful for genetic engineering ofplant cells to provide a method of controlling the timing or tissuepattern of expression of foreign DNA sequences inserted into the plantplastid genome. Constructs include those for nuclear transformationwhich provide for expression of a viral single subunit RNA polymerase inplant tissues, and targeting of the expressed polymerase protein intoplant cell plastids. Also included are plastid expression constructscomprising a promoter region which is specific to the RNA polymeraseexpressed from the nuclear expression constructs described above and aheterologous gene of interest to be expressed in the transformed plastidcells. Alternatively, the gene can be transformed/localized tochloroplast/plastid genome and expressed from there using promoters wellknown in the art (see Maliga, et al.)

4.16.5 Effects of 3′ Regions on Transgene Expression

The 3′-end regions of transgenes have been found to have a large effecton transgene expression in plants (Ingelbrecht et al., 1989). In thisstudy, different 3′ ends were operably linked to the neomycinphosphotransferase II (NptII) reporter gene and expressed in transgenictobacco. The different 3′ ends used were obtained from the octopinesynthase gene, the 2S seed protein from Arabidopsis, the small subunitof rbcS from Arabidopsis, extension form carrot, and chalcone synthasefrom Antirrhinum. In stable tobacco transformants, there was about a60-fold difference between the best-expressing construct (small subunitrbcS 3′ end) and the lowest expressing construct (shalcone synthase 3′end).

TABLE 5 PLANT PROMOTERS Promoter Reference^(a) Viral Figwort MosaicVirus (FMV) U.S. Pat. No. 5,378,619 Cauliflower Mosaic Virus (CaMV) U.S.Pat. No. 5,530,196 U.S. Pat. No. 5,097,025 U.S. Pat. No. 5,110,732 PlantElongation Factor U.S. Pat. No. 5,177,011 Tomato Polygalacturonase U.S.Pat. No. 5,442,052 Arabidopsis Histone H4 U.S. Pat. No. 5,491,288Phaseolin U.S. Pat. No. 5,504,200 Group 2 U.S. Pat. No. 5,608,144Ubiquitin U.S. Pat. No. 5,614,399 P119 U.S. Pat. No. 5,633,440 α-amylaseU.S. Pat. No. 5,712,112 Viral enhancer/Plant promoter CaMV35Senhancer/mannopine U.S. Pat. No. 5,106,739 synthase promoter ^(a)Eachreference is specifically incorporated herein by reference in itsentirety.

TABLE 6 TISSUE SPECIFIC PLANT PROMOTERS Tissue Specific PromoterTissue(s) Reference^(a) Blec epidermis U.S. Pat. No. 5,646,333 malatesynthase seeds; seedlings U.S. Pat. No. 5,689,040 isocitrate lyaseseeds; seedlings U.S. Pat. No. 5,689,040 patatin tuber U.S. Pat. No.5,436,393 ZRP2 root U.S. Pat. No. 5,633,363 ZRP2(2.0) root U.S. Pat. No.5,633,363 ZRP2(1.0) root U.S. Pat. No. 5,633,363 RB7 root U.S. Pat. No.5,459,252 root U.S. Pat. No. 5,401,836 fruit U.S. Pat. No. 4,943,674meristem U.S. Pat. No. 5,589,583 guard cell U.S. Pat. No. 5,538,879stamen U.S. Pat. No. 5,589,610 SodA1 pollen; middle layer; Van Camp etal., 1996 stomium of anthers SodA2 vasular bundles; stomata; Van Camp etal., 1996 axillary buds; pericycle; stomium; pollen CHS15 flowers; roottips Faktor et al., 1996 Psam-1 phloem tissue; cortex; Vander et al.,1996 root tips ACT11 elongating tissues and Huang et al., 1997 organs;pollen; ovules zmGBS pollen; endosperm Russell and Fromm, 1997 zmZ27endosperm Russell and Fromm, 1997 osAGP endosperm Russell and Fromm,1997 osGT1 endosperm Russell and Fromm, 1997 RolC phloem tissue; bundleGraham et al., 1997 sheath; vascular parenchyma Sh phloem tissue Grahamet al., 1997 CMd endosperm Grosset et al., 1997 Bnm1 pollen Treacy etal., 1997 rice tungro phloem Yin et al., 1997a; 1997b bacilliform virusS2-RNase pollen Ficker et al., 1998 LeB4 seeds Baumlein et al., 1991gf-2.8 seeds; seedlings Berna and Bernier, 1997 ^(a)Each reference isspecifically incorporated herein by reference in its entirety.

The ability to express genes in a tissue specific manner in plants hasled to the production of male and female sterile plants. Generally, theproduction of male sterile plants involves the use of anther-specificpromoters operably linked to heterologous genes that disrupt pollenformation (U.S. Pat. Nos. 5,689,051; 5,689,049; 5,659,124, eachspecifically incorporated herein by reference). U.S. Pat. No. 5,633,441(specifically incorporated herein by reference) discloses a method ofproducing plants with female genetic sterility. The method comprises theuse of style-cell, stigma-cell, or style- and stigma-cell specificpromoters that express polypeptides that, when produced in the cells ofthe plant, kills or significantly disturbs the metabolism, functioningor development of the cells.

TABLE 7 INDUCIBLE PLANT PROMOTERS Promoter Reference^(a) heat shockpromoter U.S. Pat. No. 5,447,858 Em U.S. Pat. No. 5,139,954 Adh1 Kyozokaet al., 1991 HMG2 U.S. Pat. No. 5,689,056 cinnamyl alcohol dehydrogenaseU.S. Pat. No. 5,633,439 asparagine synthase U.S. Pat. No. 5,595,896GST-II-27 U.S. Pat. No. 5,589,614 ^(a)Each reference is specificallyincorporated herein by reference in its entirety.4.18 Antibody Compositions and Methods of Making

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to one or more ofthe polypeptides disclosed herein. Means for preparing andcharacterizing antibodies are well known in the art (See, e.g., Harlowand Lane, 1988; incorporated herein by reference). The methods forgenerating monoclonal antibodies (mAbs) generally begin along the samelines as those for preparing polyclonal antibodies. Briefly, apolyclonal antibody is prepared by immunizing an animal with animmunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified crystal protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep, or frog cells isalso possible. The use of rats may provide certain advantages (Goding,1986, pp. 60-61), but mice are preferred, with the BALB/c mouse beingmost preferred as this is most routinely used and generally gives ahigher percentage of stable fusions.

4.19 ELISAs and Immunoprecipitation

ELISAs may be used in conjunction with the invention. The production anduse of ELISAs or kits emplyoying such ELISAs are well know to those ofskill in the art.

4.20 Western Blots

The compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-peptide antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

4.21 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated crystal proteins are contemplated to be usefulfor increasing the insecticidal activity of the protein, andconsequently increasing the insecticidal activity and/or expression ofthe recombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 8.

TABLE 8 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8tryptophan(−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate(−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine(−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

5.0 EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

5.1 Example 1 Isolation of B. thuringiensis Strains EG4550 and EG5899

Crop dust samples were obtained from various sources throughout theUnited States and abroad, typically from grain-storage facilities. Thecrop dust samples were treated and spread on agar plates to isolateindividual Bacillus-type colonies, e.g., B. thuringiensis, as describedin U.S. Pat. No. 5,187,091, specifically incorporated herein byreference in its entirety. Phase-contrast microscopy was used tovisually identify cells with crystalline inclusions in the colonies thatgrew after this treatment. Crystal-producing strains were thencharacterized by modified Eckhardt agarose gel electrophoresis asdescribed by Gonzalez et al., (1982). This procedure allows thevisualization of the array of native plasmids in a B. thuringiensisstrain. The plasmid arrays can be compared to those of known serovars ofB. thuringiensis to facilitate the identification of wild-type strains(Carlton and Gonzalez, 1985).

Strain EG4550 is a crystal-producing B. thuringiensis strain isolatedfrom a New York crop dust sample. The crystalline inclusions ofsporulated EG4550 have a distinct morphology and resemble tiny rods. Theplasmid array of EG4550 does not resemble the array of any of the knownserovars of B. thuringiensis.

Strain EG5899 is a crystal-producing B. thuringiensis strain isolatedfrom a California crop dust sample. The crystalline inclusions ofsporulated EG5899 are unusual in that they appear to be multipleattached crystals with an irregular morphology. The plasmid array ofEG5899 does not resemble the array of any of the known serovars of B.thuringiensis.

Insect bioassay of the B. thuringiensis strains EG4550 and EG5899indicated that these strains are toxic to larvae of coleopteran insects,including SCRW, suggesting that the crystals in these strains containednovel insecticidal proteins. EG4550 and EG5899 were deposited with theARS Patent Culture Collection and been assigned NRRL numbers B-21784 andB-21783, respectively. These strains and other strains of the presentinvention are listed in Table 9:

TABLE 9 WILD-TYPE AND RECOMBINANT BACTERIAL STRAINS^(a) Plasmid NRRLNRRL Deposit cry Gene(s) Antibiotic Strain Accession # Date^(b) OrganismPlasmid Cloned Insert Vector Present Marker EG4550 B-21784 May 30, 1997B. thuringiensis — — — cryET39, 74, 75 — EG5899 B-21783 May 30, 1997 B.thuringiensis — — — cryET39, 74, 75 — EG11582 — — E. coli pEG1337 8.4-kbHindIII pUC18 cryET39, 74, 75 Amp EG11525 — — E. coli pEG1321 8.4-kbHindIII pEG597 cryET39, 74, 75 Amp EG11529 B-21917 Feb. 12, 1998 B.thuringiensis pEG1321 8.4-kb HindIII pEG597 cryET39, 74, 75 Cm EG11521 —— E. coli pEG1319 8.4-kb HindIII pBluescript cryET39, 74, 75 Amp EG11934— — B. thuringiensis pEG1918 4.5-kb HindIII pHT315 cryET75 Eryth EG11935— — B. thuringiensis pEG1919 3.2-kb HindIII- pHT315 cryET74 Eryth EcoRIEG11936 — — B. thuringiensis pEG1920 3.7-kb HindIII pHT315 cryET39, 74Eryth EG11937 — — B. thuringiensis pEG1921 1.4-kb HindIII pEG1915cryET39 Eryth EG4100 B-21786 May 30, 1997 B. thuringiensis — — — cryET69— EG11647 B-21787 May 30, 1997 B. thuringiensis pEG1820 MboI partialpHT315 cryET69 Eryth EG9444 B-21785 May 30, 1997 B. thuringiensis — — —cryET71, 79 — EG11648 B-21788 May 30, 1997 B. thuringiensis pEG1821 MboIpartial pHT315 cryET71, 79 Eryth EG4851 B-21915 Feb. 12, 1998 B.thuringiensis — — — cryET76, 80, 84 — EG11658 B-21916 Feb. 12, 1998 B.thuringiensis pEG1823 MboI partial pHT315 cryET76, 80, 84 Eryth ^(a)Thedetails of the construction of the strains listed above are included inthe following Examples. ^(b)The subject cultures have been depositedunder conditions that assure that access to the cultures will beavailable during the pendency of this patent application to onedetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 C.F.R. §1.14 and 35 U.S.C. §122. The deposits areavailable as required by foreign patent laws in countries whereincounterparts of the subject application, or its progeny, are filed.However, it should be understood that the availability of a deposit doesnot constitute a license to practice the subject invention in derogationof patent rights granted by governmental action. The subject culturedeposits will be stored and made available to the public in accord withthe provisions of the Budapest Treaty for the Deposit of Microorganisms,i.e., they will be stored with all the care necessary to keep themviable and uncontaminated for a period of at least five years after themost recent request for the finishing of a sample of the deposit, and inany case, for a period of at least 30 (thirty) years after the date ofdeposit or for the enforceable life of any patent which may issuedisclosing the cultures. The depositor acknowledges the duty to replacethe deposits should the depository be unable to furnish a sample whenrequested, due to the condition of the deposits. All restrictions on theavailability to the public of the subject culture deposits will beirrevocably removed upon the granting of a patent disclosing them.Cultures shown in Table 3 were deposited in the permanent collection ofthe Agricultural Research Service Culture Collection, Nothern RegionalResearch Laboratory (NRRL), located at 1815 N. University Street,Peoria, IL 61604, under the terms of the Budapest Treaty.

5.2 Example 2 Evaluation of the Crystal Proteins of EG4550 and EG5899

Strains EG4550 and EG5899 were grown in C2 sporulation medium (Donovan,et al., J. Biol. Chem., 263:561-567, 1988) for three days at 30° C.during which the cultures grew to stationary phase, sporulated andlysed, releasing the protein inclusions into the medium. The cultureswere centrifuged to harvest cell pellets containing the spores andcrystals. The pellets were washed by suspension in a solution of 0.005%Triton X-100® and centrifuged. The washed pellets were resuspended atone-tenth the original volume in 0.005% Triton X-100®.

Crystal proteins were solubilized from the spores-crystals suspensionsby incubating in solubilization buffer [0.14 M Tris-HCl pH 8.0, 2%(wt./vol.) sodium dodecyl sulfate (SDS), 5% (vol./vol.)2-mercaptoethanol, 10% (vol./vol.) glycerol, and 1% bromphenol blue] at100° C. for 5 min. The solubilized crystal proteins weresize-fractionated by SDS-PAGE using a gel with an acrylamideconcentration of 12.5%.

After size fractionation the proteins were visualized by staining withCoomassie Brilliant Blue R-250. Strain EG4550 displayed proteins withapproximate molecular weights of 45 and 15 kDa. Strain EG5899 displayedproteins of approximate molecular weights of 160 kDa, 45 kDa, 35 kDa,and 15 kDa.

5.3 Example 3 Characterization of the CryET39 Crystal Protein of EG4550

The NH₂-terminal sequence of the approximately 45-kDa protein of EG4550,designated CryET39, was determined. A sporulated culture of EG4550 waswashed and resuspended. The crystal proteins in the suspension weresolubilized and run on a 10% acrylamide gel following the procedures forSDS-PAGE analysis. After electrophoresis the proteins were transferredto a BioRad PVDF membrane using standard western blotting procedures.Following transfer the membrane was rinsed 3× in distilled H₂O andstained for 1 min using Amido Black 1013 (Sigma Chemical Co., St. Louis,Mo.). The filter was destained for 1 min in 5% acetic acid and thenrinsed in 3 changes of distilled H₂O. The portion of the filtercontaining the approximately 45-kDa CryET39 band was excised with arazor blade. This procedure resulted in a pure form of CryET39 beingobtained as a protein band blotted onto a PVDF membrane (BioRad,Hercules, Calif.).

The determination of the NH₂-terminal amino acid sequence of thepurified CryET39 protein immobilized on the membrane was performed inthe Department of Physiology at the Tufts Medical School, Boston, Mass.using standard Edman degradation procedures. The NH₂-terminal sequencewas determined to be:

(SEQ ID NO:20) 1   2   3   4   5   6   7   8   9   10  11  12 Met LeuAsp Thr Asn Lys Val Tyr Glu Ile Ser Asn 13  14  15 His Ala Asn

Computer algorithms (Korn and Queen, 1984) were used to compare theNH₂-terminal sequence of the CryET39 protein with the amino acidsequences of all B. thuringiensis crystal proteins of which theinventors were aware including the sequences of all B. thuringiensiscrystal proteins which had been published in scientific literature,international patent applications, or issued patents. A list of thecrystal proteins whose sequences have been published and assigned agene/protein designation is shown in Table 2.

5.4 Example 4 Isolation of a DNA Fragment Comprising the CryET39 Gene

In order to identify the gene encoding CryET39, an oligonucleotide probespecific for the NH₂-terminal amino acid sequence of the protein wasdesigned. Using codons typically found in B. thuringiensis toxin genesan oligonucleotide of 41 nucleotides was synthesized by Integrated DNATechnologies, Inc. (Coralville, Iowa) and designated wd271. The sequenceof wd271 is:

(SEQ ID NO:21) 5′-ATGTTAGATACAAATAAAGTATATGAAATTTCAAATCATGC-3′

Radioactively labeled wd271 was then used as a probe in Southernhybridization experiments, as described below, to identify a restrictionfragment containing the cryET39 gene. Total DNA was extracted fromstrains EG4550 and EG5899 by the following procedure. Vegetative cellswere resuspended in a lysis buffer containing 50 mM glucose, 25 mMTris-HCl (pH 8.0), 10 mM EDTA, and 4 mg/ml lysozyme. The suspension wasincubated at 37° C. for one hour. Following incubation, the suspensionwas extracted once with an equal volume of phenol, then once with anequal volume of phenol:chloroform:isoamyl alcohol (25:24:1), and oncewith an equal volume of chloroform:isoamyl alcohol (24:1). The DNA wasprecipitated from the aqueous phase by the addition of one-tenth volume3 M sodium acetate then two volumes 100% ethanol. The precipitated DNAwas collected by centrifugation, washed with 70% ethanol and resuspendedin dH₂O.

The extracted DNA was then digested, in separate reactions, with variousrestriction endonucleases, including EcoRI and HindIII, using conditionsrecommended by the manufacturer (Promega Corp., Madison, Wis.). Thedigested DNA was size-fractionated by electrophoresis through a 0.8%agarose gel in 1×TBE (0.089 M Tris-borate, 0.089 M boric acid, 0.002 MEDTA) overnight at 2 volts/cm of gel length. The fractionated DNAfragments were then transferred to a Millipore Immobilon-NC®nitrocellulose filter (Millipore Corp., Bedford, Mass.) according to themethod of Southern (1975). The DNA fragments were fixed to thenitrocellulose by baking the filter at 80° C. in a vacuum oven.

Identification of the DNA fragment(s) containing the sequence encodingthe NH₂-terminus of the CryET39 protein (see Example 3) was accomplishedby using the oligonucleotide wd271 as a hybridization probe. Toradioactively label the probe, 1 to 5 pmoles wd271 was added to areaction containing [γ-³²P]ATP (3 μl of 3,000 Ci/mmole at 10 mCi/ml in a20-μl reaction volume), a 10× reaction buffer (700 mM Tris-HCl (pH 7.8),100 mM MgCl₂, 50 mM DTT), and 10 units T4 polynucleotide kinase (PromegaCorp.). The reaction was incubated 20 min at 37° C. to allow thetransfer of the radioactive phosphate to the 5′-end of theoligonucleotide, thus making it useful as a hybridization probe.

The labeled probe was then incubated with the nitrocellulose filterovernight at 45° C. in 3×SSC, 0.1% SDS, 10× Denhardt's reagent (0.2%BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll®), 0.2 mg/ml heparin.Following incubation the filter was washed in several changes of 3×SSC,0.1% SDS at 45° C. The filter was blotted dry and exposed to KodakX-OMAT AR X-ray film (Eastman Kodak Company, Rochester, N.Y.) overnightat −70° C. with a DuPont Cronex Lightning Plus screen to obtain anautoradiogram.

Examination of the autoradiogram identified an approximately 2.5-kbwd271-hybridizing EcoRI fragment in DNA from strain EG4550. StrainEG5899 had an approximately 8.4-kb HindIII restriction fragment thatspecifically hybridized to the labeled wd271. This result indicated thatboth EG4550 and EG5899 contained related, or perhaps identical, copiesof the cryET39 gene.

5.5 Example 5 Cloning of the CryET39 Gene

The first cloning study included the isolation of the 2.5-kb EcoRIfragment of EG4550 in order to express and characterize the CryET39protein of EG4550. When this fragment was cloned and expressed in a B.thuringiensis recombinant strain, however, only the 15 kDa protein wasproduced, indicating that the 2.5-kb EcoRI fragment does not contain acomplete and functional cryET39 gene. This result also indicated thatthe genes for the 15-kDa crystal protein and CryET39 were, however, inclose proximity. The recombinant B. thuringiensis strain expressing the15-kDa protein, designated EG11467, was not toxic to larvae of SCRW.

The approximately 8.4 kb HindIII restriction fragment containing thecryET39 gene from EG5899 was isolated from total genomic DNA asdescribed in Section 5.4. The DNA was digested with HindIII andelectrophoresed through a 0.8% agarose, 1×TBE gel, overnight at 2volts/cm of gel length. The gel was stained with ethidium bromide sothat the digested DNA could be visualized when exposed to long-wave UVlight. Gel slices containing DNA fragments of approximately 8.0-9.0 kbwere excised from the gel with a razor blade. The DNA fragments werethen purified from the gel slice using the Geneclean® procedure (Bio101, Vista, Calif.).

The isolated DNA fragments were ligated into the phagemid pBluescript®II SK+ (Stratagene, LaJolla, Calif.) to create a library in E. coli ofsize-selected HindIII restriction fragments. The phagemid DNA vectorpBluescript® II SK+ can replicate at a high copy number in E. coli andcarries the gene for resistance to the antibiotic ampicillin, which canbe used as a selectable marker. The fragments were mixed withHindIII-digested pBluescript® II SK+ that had been treated withbacterial alkaline phosphatase (GibcoBRL, Gaithersburg, Md.) to removethe 5′ phosphates from the digested plasmid to prevent re-ligation ofthe vector to itself. T4 ligase and a ligation buffer (Promega Corp.)were added to the reaction containing the digested phagemid and thesize-selected HindIII fragments. These were incubated at roomtemperature for 1 hour to allow the insertion and ligation of theHindIII fragments into the pBluescript® II SK+ vector.

The ligation mixture was introduced into transformation-competent E.coli DH5α™ cells (GibcoBRL) following procedures described by themanufacturer. The transformed E. coli cells were plated on LB agarplates containing 50 μg/ml ampicillin and incubated overnight at 37° C.The growth of several hundred ampicillin-resistant colonies on eachplate indicated the presence of the recombinant plasmid in the cells ofeach of those colonies.

To isolate the colonies harboring the cloned 8.4-kb HindIII fragmentcontaining the cryET39 gene, colonies were first transferred tonitrocellulose filters. This was accomplished by placing a circularfilter (Millipore HATF 085 25, Millipore Corp., Bedford, Mass.) directlyon top of the LB-ampicillin agar plates containing the transformedcolonies. When the filter was slowly peeled off of the plate thecolonies stuck to the filter giving an exact replica of the pattern ofcolonies from the original plate. Enough cells from each colony wereleft on the plate that 5 to 6 hours of growth at 37° C. restored thecolonies. The plates were then stored at 4° C. until needed. Thenitrocellulose filters with the transferred colonies were then placed,colony-side up, on fresh LB-ampicillin agar plates and allowed to growat 37° C. until the colonies reached an approximate diameter of 1 mm.

To release the DNA from the recombinant E. coli cells the nitrocellulosefiters were placed colony-side up on 2 sheets of Whatman 3 MMChromatography paper (Whatman International Ltd., Maidstone, England)soaked with 0.5 N NaOH, 1.5 M NaCl for 15 min. This treatment lysed thecells and denatured the released DNA allowing it to stick to thenitrocellulose filter. The filters were then neutralized by placing thefilters, colony-side up, on 2 sheets of Whatman paper soaked with 1 Mammonium acetate, 0.02 M NaOH for 10 min. The filters were then rinsedin 3×SSC, air dried, and baked for 1 hour at 80° C. in a vacuum oven toprepare them for hybridization.

The NH₂-terminal oligonucleotide specific for the cryET39 gene, wd271,was labeled at the 5′ end with γ-³²P and T4 polynucleotide kinase asdescribed above. The labeled probe was added to the filters in 3×SSC,0.1% SDS, 10× Denhardt's reagent (0.2% BSA, 0.2% polyvinylpyrrolidone,0.2% Ficoll®), 0.2 mg/ml heparin and incubated overnight at 40° C. Theseconditions were chosen to allow hybridization of the labeledoligonucleotide to related sequences present on the nitocellulose blotsof the transformed E. coli colonies. Following incubation the filterswere washed in several changes of 3×SSC, 0.1% SDS at 45° C. The filterswere blottted dry and exposed to Kodak X-OMAT AR X-ray film (EastmanKodak) overnight at −70° C. with a DuPont Cronex Lightning Plus screen.

Several colonies from this transformation hybridized to wd271. Thesecolonies were identified by lining up the signals on the autoradiogramwith the colonies on the original transformation plates. The isolatedcolonies were then grown in LB-ampicillin liquid medium from which thecells could be harvested and recombinant plasmid prepared by thestandard alkaline-lysis miniprep procedure (Maniatis et al., 1982). Theisolated plasmids were digested with the restriction enzyme HindIIIwhich indicated that the cloned fragments of EG5899 DNA were of theexpected size, i.e. 8.4-kb. HindIII-digested plasmid DNA from six of thehybridizing colonies was electrophoresed through an agarose gel andtransferred to nitrocellulose as described above. The blot was thenhybridized with the oligonucleotide wd271 that had been radioactivelylabeled at the 5′ end with γ-³²P and T4 polynucleotide kinase. Theapproximately 8.4-kb insert fragments from all six of the digestedplasmids hybridized with wd271 confirming the presence of the cryET39gene. One of the plasmids with the 8.4 kb insert containing the cryET39gene was designated pEG1319. The E. coli strain containing pEG1319 hasbeen designated EG11521.

5.6 Example 6 Expression of Recombinant Proteins from EG11529

To characterize the properties of the CryET39 protein it was necessaryto express the cloned cryET39 gene in B. thuringiensis cells that do notproduce any crystal proteins (Cry⁻). To accomplish this, the cloned8.4-kb HindIII fragment from pEG1319 was inserted into a plasmid capableof replicating in B. thuringiensis, thus allowing the expression of thecryET39 gene and production of the encoded protein.

pEG1319 was digested with HindIII to excise the cloned 8.4-kb fragment.The digested plasmid was resolved on an agarose gel and a slice of thegel containing the 8.4-kb fragment was excised. The 8.4-kb Hind IIIfragment was purified from the gel slice using the GeneClean procedure(Bio101). The fragment was ligated into a B. thuringiensis/E. colishuttle vector that had been digested with HindIII and treated withbacterial alkaline phosphatase. This shuttle vector, designated pEG597,was described by Baum et al., (1990). pEG597 is capable of replicationin both E. coli and B. thuringiensis, conferring ampicillin resistanceto E. coli and chloramphenicol resistance to B. thuringiensis. Theligation mixture was introduced into E. coli DH5α™ cells usingtransformation procedures described by the manufacturer (GibcoBRL).Plasmid DNA was prepared from Amp^(R) transformants and restrictionenzyme analysis was performed to confirm the proper construction. Aplasmid containing the 8.4-kb HindIII fragment inserted into the pEG597vector was designated pEG1321. The E. coli strain harboring pEG1321 wasdesignated EG11525.

pEG1321 was introduced into a Cry⁻ B. thuringiensis strain, EG10368, byelectroporation (Macaluso and Mettus, 1991). Cells transformed tochloramphenicol resistance were selected by incubation overnight on LBagar plates containing 3 μg/ml chloramphenicol. Plasmid DNA was isolatedfrom several of the B. thuringiensis transformants. The isolated plasmidwas digested with HindIII and electrophoresed through an agarose gel.All of the transformants had restriction fragments corresponding to the8.4 kb cryET39 fragment and the pEG597 vector. To verify the correctplasmid construction the restriction fragments were blotted to anitrocellulose filter which was then hybridized with thecryET39-specific oligo wd271, as described above. The wd271probehybridized to the cloned 8.4 kb HindIII fragments confirming thatpEG1321 contains the cryET39 gene and that it had been successfullyintroduced into B. thuringiensis. The B. thuringiensis recombinantstrain containing pEG1321 was designated EG11529. EG11529 was depositedwith the NRRL and given the accession number B-21917.

EG11529 was grown in DSM+glucose sporulation medium containing 5 μg/mlchloramphenicol [0.8% (wt./vol.) Difco nutrient broth, 0.5% (wt./vol.)glucose, 10 mM K₂HPO₄, 10 mM KH₂PO₄, 1 mM Ca(NO₃)₂, 0.5 mM MgSO₄, 10 μMMnCl₂, 10 FeSO₄] for three days at 30° C. during which the culture grewto stationary phase, sporulated and lysed, thus releasing the proteininclusions into the medium. The cultures were harvested bycentrifugation. The pellet consisting of spores and protein crystals waswashed in a solution of 0.005% Triton X-100®, 2 mM EDTA and centrifuged.The washed pellet was suspended at one-tenth the original volume in0.005% Triton X-100®, 2 mM EDTA.

Crystal proteins were solubilized from the spores-crystal suspension byincubating the suspension in solubilization buffer [0.14 M Tris-HCl pH8.0, 2% (wt./vol.) sodium dodecyl sulfate (SDS), 5% (vol./vol.)2-mercaptoethanol, 10% (vol./vol.) glycerol, and 0.1% bromphenol blue]at 100° C. for 5 min. The solubilized crystal proteins weresize-fractionated by SDS-PAGE. After size fractionation the proteinswere visualized by staining with Coomassie Brilliant Blue R-250. Thisanalysis showed that three distinct crystal proteins were produced instrain EG11529. In addition to the 44-kDa CryET39 toxin, approximately15- and 35-kDa polypeptides were also produced.

The 35-kDa crystal protein expressed in B. thuringiensis EG11529 couldbe separated from the 44-kDa (CryET39) and 15-kDa proteins bycentrifugation through a sucrose step gradient (steps: 55%, 68%, 72%,79%) as described in Section 5.12. Determination of the NH₂-terminalamino acid sequence of the isolated 35-kDa protein was accomplishedusing procedures described in Section 5.3. The NH₂-terminal amino acidsequence of the 35-kDa protein was shown to be:

SILNLQDLSQKYMTAALNKI (SEQ ID NO:22)

Comparison of the NH₂-terminus of the 35-kDa protein with the deducedamino acid sequence of CryET39 confirmed that it was not a processedform of the CryET39 protein. The approximately 35 kDa protein wasdesignated CryET75, and the gene encoding it (which resides on the8.4-kb fragment from EG5899) was designated cryET75.

The sucrose gradient fraction containing CryET39 also contained theapproximately 15-kDa protein, designated CryET74. The NH₂-terminal aminoacid sequence of CryET74 was determined as described for CryET39 inSection 5.3. The NH₂-terminal amino acid sequence of the isolatedCryET74 protein was determined to be:

SARQVHIQINNKTRH (SEQ ID NO:23)

Comparison of this sequence with that of CryET39 and CryET75 showed thatCryET74 was a unique protein encoded by a third gene, designatedcryET74, that was contained on the 8.4-kb HindIII fragment cloned fromEG5899.

5.7 Example 7 Sequencing of the Cry Genes and Determination of the AminoAcids Sequences of the Encoded Polypeptides

To facilitate the sequencing of the cryET39, cryET74, and cryET75 genes,the 8.4-kb HindIII fragment of pEG1319 was subcloned intoHindIII-digested pUC18 (Yanisch-Perron et al., 1985). This plasmid wasdesignated pEG1337, and is shown in FIG. 1.

Preparation of pEG1337 double-stranded plasmid template DNA wasaccomplished using either a standard alkaline lysis procedure or aQiagen Plasmid Kit (Qiagen Inc., Chatworth, Calif.) following themanufacturer's procedures. The sequencing reactions were performed usingthe Sequenase™ Version 2.0 DNA Sequencing Kit (United StatesBiochemical/Amersham Life Science Inc., Cleveland, Ohio) following themanufacturer's procedures and using ³⁵S-[dATP] as the labeling isotope(DuPont NEN Research Products, Boston, Mass.). Denaturing gelelectrophoresis of the reactions was performed on a 6% (wt./vol.)acrylamide, 42% (wt./vol.) urea sequencing gel. The dried gel wasexposed to Kodak X-OMAT AR X-ray film (Eastman Kodak) overnight at roomtemperature.

The NH₂-terminal specific oligonucleotide wd271 was used as the initialsequencing primer. The entire sequence for the cryET39 gene wasdetermined using the procedures described above. Successiveoligonucleotides to be used for priming sequencing reactions weredesigned from the sequencing data of the previous set of reactions. Inthis way the DNA sequencing progressed along both the top and bottomstrand of the cryET39 gene in a step-wise fashion.

An oligonucleotide primer based on the NH₂-terminal amino acid sequenceof the CryET75 protein was designed for use in sequencing the cryET75gene. The oligonucleotide was designated MR51 and had the sequence:

5′-TCACAAAAATATATGAACAGC-3′ (SEQ ID NO:24)

Using the DNA sequencing procedures described above, a partialnucleotide sequence of the cryET75 gene was determined, with thecompletion of the sequence being achieved using automated sequencing.DNA samples were sequenced using the ABI PRISM® DyeDeoxy sequencingchemistry (Applied Biosystems, Inc., CA) according to the manufacturer'sprotocol. The completed reactions were run on an ABI 377 automated DNAsequencer. DNA sequence data were analyzed using Sequencher v3.0 DNAanalysis software (Gene Codes Corporation, Ann Arbor, Mich.). The aminoacid sequence of the CryET75 protein was then derived by translating theopen reading frame of cryET75. The determined NH₂-terminal sequence ofCryET75 was identical with the NH₂-terminal amino acid sequence derivedfrom the nucleotide sequence.

Studies in which the 8.4-kb HindIII fragment from EG11529 was furtherdigested and the fragments sub-cloned to express the crystal proteingenes individually, or in combination, are described in Section 5.11.The expression of the CryET39 protein was dependent on cloning thecryET74 gene on the same restriction fragment. This suggested that thecryET74 gene was located upstream of the cryET39 gene and that thepromoter for cryET74 also directs the expression of cryET39.Oligonucleotides specific for the DNA sequence 5′ to the beginning ofthe cryET39 gene were designed for use as primers for automatedsequencing. Successive primers were designed based on the data derivedfrom each set of sequencing reactions. In this way the region upstreamof cryET39 was sequenced in a step-wise fashion. A translation of theDNA sequence revealed an open reading frame encoding the CryET74protein. Examination of the derived amino acid sequence found a regionidentical to the determined NH₂-terminal amino acid sequence of CryET74,identifying the open reading frame as the cryET74 gene.

5.7.1 CryET39

The DNA sequence of the CryET39 gene is represented by SEQ ID NO:7, andencodes the amino acid sequence of the CryET39 polypeptide, representedby SEQ ID NO:8.

5.7.1.3 Characteristics of the CryET39 Polypeptide Isolated from EG5899

The CryET39 polypeptide comprises a 385-amino acid sequence, has acalculated molecular mass of 44,246 Da, and has a calculated isoelectricconstant (pI) equal to 5.47. The amino acid composition of the CryET39polypeptide is given in Table 11.

TABLE 11 AMINO ACID COMPOSITION OF CRYET39 Amino Acid # Residues % TotalAla 6 1.5 Arg 3 0.7 Asn 31 8.0 Asp 23 5.9 Cys 2 0.5 Gln 17 4.4 Glu 277.0 Gly 19 4.9 His 8 2.0 Ile 32 8.3 Leu 33 8.5 Lys 39 10.1 Met 8 2.0 Phe6 1.5 Pro 16 4.1 Ser 30 7.7 Thr 36 9.3 Trp 7 1.8 Tyr 24 6.2 Val 18 4.6Acidic (Asp + Glu) 50 Basic (Arg + Lys) 42 Aromatic (Phe + Trp + Tyr) 37Hydrophobic (Aromatic + Ile + Leu + Met + Val) 1265.7.2 CryET74

The DNA sequence of the CryET74 gene is represented by SEQ ID NO:5, andencodes the amino acid sequence of the CryET74 polypeptide, representedby SEQ ID NO:6.

5.7.2.3 Characteristics of the CryET74 Polypeptide

The CryET74 polypeptide comprises a 119-amino acid sequence, has acalculated molecular mass of 13,221 Da, and has a calculated pI equal to6.21. The amino acid composition of the CryET74 polypeptide is given inTable 12.

TABLE 12 AMINO ACID COMPOSITION OF CRYET74 Amino Acid # Residues % TotalAla 4 3.3 Arg 6 5.0 Asn 6 5.0 Asp 7 5.8 Cys 1 0.8 Gln 3 2.5 Glu 8 6.7Gly 10 8.4 His 5 4.2 Ile 9 7.5 Leu 6 5.0 Lys 7 5.8 Met 2 1.6 Phe 4 3.3Pro 3 2.5 Ser 13 10.9 Thr 12 10.0 Trp 1 0.8 Tyr 4 3.3 Val 8 6.7 Acidic(Asp + Glu) 15 Basic (Arg + Lys) 13 Aromatic (Phe + Trp + Tyr) 9Hydrophobic (Aromatic + Ile + Leu + Met + Val) 345.7.3 CryET75

The DNA sequence of the CryET75 gene is represented by SEQ ID NO:15, andencodes the amino acid sequence of the CryET75 polypeptide, representedby SEQ ID NO:16.

5.7.3.3 Characteristics of the CryET75 Polypeptide

The CryET75 polypeptide comprises a 310-amino acid sequence, has acalculated molecular mass of 34,259 Da, and has a calculated pI equal to5.67. The amino acid composition of the CryET75 polypeptide is given inTable 13.

TABLE 13 AMINO ACID COMPOSITION OF CRYET75 Amino Acid # Residues % TotalAla 15 4.8 Arg 5 1.6 Asn 15 4.8 Asp 17 5.4 Cys 2 0.6 Gln 11 3.5 Glu 227.0 Gly 17 5.4 His 6 1.9 Ile 22 7.0 Leu 24 7.7 Lys 29 9.3 Met 7 2.2 Phe11 3.5 Pro 9 2.9 Ser 34 10.9 Thr 33 10.6 Trp 1 0.3 Tyr 11 3.5 Val 19 6.1Acidic (Asp + Glu) 39 Basic (Arg + Lys) 34 Aromatic (Phe + Trp + Tyr) 23Hydrophobic (Aromatic + Ile + Leu + Met + Val) 95

5.8 Example 8 Homology Analyses for CryET39

The deduced amino acid sequence of the CryET39 protein was used to queryelectronic sequence databases for related protein homologies. TheSWISS-PROT ALL (swall) database was queried using FASTA version 3.15(Pearson and Lipman, 1988) on the FASTA server at the EuropeanBioinformatics Institute under the following parameters (matrix=pam150,ktup=2, gapcost=−12, gapxcost=−2). The results of the database searchshowed that CryET39 exhibited ˜25% amino acid sequence identity over a322-amino acid region of the 42-kDa mosquitocidal crystal protein fromB. sphaericus. CryET39 also showed ˜20% sequence identity over a 343amino acid region of the 51-kDa crystal protein from B. sphaericus. Noother protein sequences in the database showed any significant sequencesimilarity with the CryET39 sequence. The amino acid sequence of CryET39was also used to query the non-redundant (nr) database of the NationalCenter Biotechnology Information (NCBI) using BLASTP version 2.0(Altschul et al., 1997) using the following parameters: matrix=blosum62,gapped alignment, other parameters=default settings. The nr databasecomprises sequence entries from PDB, SWISS-PROT, PIR, and CDStranslations of GenBank. The results of this search were in agreementwith those obtained using the FASTA search.

5.9 Example 9 Database Searches for CryET74-Related Proteins

The deduced amino acid sequence for CryET74 was also used to query theSWISS-PROT ALL and nr databases using FASTA and BLASTP as described inSection 5.8. No proteins were found showing any significant sequencesimilarity to CryET74.

5.10 Example 10 Database Searches for CryET75-Related Proteins

The deduced amino acid sequence for CryET75 was also used to query theSWISS-PROT ALL and nr databases using FASTA and BLASTP as described inSection 5.8. The FASTA search revealed that CryET75 showed a 28.1%sequence identity with Cry15Aa (Genbank Accession Number M76442) over a121-amino acid region. The BLASTP analysis revealed 23% sequenceidentity over a 231-amino acid region.

5.11 Example 11 Subcloning and Expression of the CryET39 and CryET74Genes

The sucrose gradient fraction of parasporal crystals obtained from lysedcultures of strain EG11529 contained both CryET39 and CryET74polypeptides. Bioassay evaluation of the CryET39 and CryET74 preparationdemonstrated that this preparation was as toxic to WCRW larvae as totalcrystal protein prepared from EG11529. To determine the insecticidalactivity of the CryET39 protein alone it was necessary to clone thecryET39 gene downstream from another promoter. As described below, thiswas accomplished by using the PCR™, to amplify the promoter region forthe B. thuringiensis crystal protein, Cry2Ac (Wu et al., 1991) andplacing it upstream of a PCR™-amplified cryET39 gene in a shuttlevector, thus allowing for the expression of only the CryET39 protein ina recombinant B. thuringiensis strain.

Oligonucleotides were designed for use as primers in the PCR™amplification and subsequent cloning of the regulatory region of thecry2Ac gene; including the open reading frames ORF1 and ORF2, theribosome binding site, and the start codon for Cry2Ac. Oligo mr47includes the EcoRI restriction site 2124 base pairs upstream from thestart codon of the cry2Ac coding region. The sequence of mr47 is:

(SEQ ID NO:25) 5′-ATATCTATA GAATTC GCAATTCGTCCATGTG-3′             EcoRI

The complementary oligonucleotide primer, mr43, consists of the invertedcomplementary sequence for the ribosome binding site and start codon(Met) for the cry2Ac gene. A HindIII site has been incorporated betweenthe RBS and the Met codon to allow for an in frame insertion of thesequence of the cryET39 gene. The sequence of mr43 is:

(SEQ ID NO:26) 5′-CAGTATTCATATAAGCTT CCTCCTTTAATA-3′          Met  HindIII RBS

The PCR™ reaction to amplify the cryET39 gene consisted of thefollowing: four deoxynucleosidetriphosphates—dATP, dTTP, dCTP, dGTP—at afinal concentration of 200 μM; 5 μl 10×Taq Extender™ Buffer (StratageneCloning Systems) for a final concentration of 1×; 10 ng pEG1273, whichconsisted of pUC18 into which the cry2Ac gene has been cloned; theoligonucleotide primers mr47 and mr43 at a final concentration of 2.5 μMeach; 2.5 units Taq Extender™ (Stratagene Cloning Systems); 2.5 unitsTaq Polymerase (Promega Corp.); and dH₂O to a final reaction volume of50 μl. The reaction was performed in a PowerBlock™ EasyCycler™ Seriestemperature cycler (Ericomp, Inc., San Diego, Calif.). Cyclingconditions consisted of a 2-min denaturation step at 94° C., followed by30 cycles of 94° C. for 1 min, annealing at 50° C. for 1 min, andextension at 72° C. for 2 min. Following the cycling 5 μl of thereaction was electrophoresed through a 0.8% agarose gel to verify thatan approximately 2-kb product band was produced by the PCR™. Theremainder of the reaction product was purified using a QIAquick™ spincolumn following the manufacturer's instructions (QIAGEN, Inc.).

The PCR™-amplified cry2Ac promoter was then cloned into the E. coli/B.thuringiensis shuttle vector pHT315 (Arantes and Lereclus, 1991). Thiswas accomplished by digesting both pHT315 and the PCR™ product, inseparate reactions, with the restriction enzymes EcoRI and HindIII.These enzymes cut within the multiple cloning region of pHT315 and nearthe ends of the PCR™ product, within the sequences specified by theoligonucleotides mr47 and mr43. The digested PCR™ product was isolatedby running the reaction through an agarose gel, followed by purificationof the approximately 2-kb fragment using the Geneclean® procedure (Bio101). Digested pHT315 was purified in a similar manner. The fragment wasthen ligated into the digested pHT315 in a reaction containing T4 DNAligase and a ligation buffer (Promega Corp.).

The ligation mixture was introduced into transformation-competent E.coli DH5α™ cells (GibcoBRL) following procedures described by themanufacturer. The E. coli cells were plated on LB agar plates containing50 μg/ml ampicillin and incubated overnight at 37° C. pHT315 contains agene that confers ampicillin resistance to recombinant cells into whichit has been successfully introduced. Plasmid DNA was prepared fromseveral ampicillin-resistant clones and digested with EcoRI and HindIIIto confirm the presence of the 2-kb insert. One of these plasmids,designated pEG1915, was used for the cloning and expression of thecryET39 gene.

PCR™ was used to amplify cryET39 from the cloned 8.4-kb HindIII fragmentin pEG1337. Oligonucleotide primers were designed to facilitate theinsertion of cryET39 into pEG1915 so that the gene could be expressedfrom the cry2Ac promoter. The cryET39-specific oligonucleotide, mr44,includes the start codon (Met) for cryET39 with a HindIII siteengineered 5′ to the start codon. The sequence of mr44 is:

(SEQ ID NO:27) 5′-AAGGTGAAGCTTTTATGTTAGATACTAATAAAGTTTATG-3′          Hind III   Met

A second primer, designated mr45, was designed to be complementary to asequence 212 base pairs 3′ to the end of the cryET39 coding region. AHindIII site was incorporated into the sequence of mr45.

5′-CCGGAATAGAAGCTTTGCATATGG-3′ (SEQ ID NO:28)             HindIII

The cryET39 PCR™ product generated using mr44 and mr45 as a primers wascut with HindIII and inserted into the HindIII site specified by mr43 inthe plasmid pEG1915. This places the Met codon of the cryET39 gene7-base pairs downstream from the ribosome binding site of the clonedcry2Ac promoter. Such a configuration was expected to allow theefficient expression of the recombinant CryET39 protein. The ligationreaction that was performed to insert the cryET39 gene into pEG1915 wasused to transform E. coli DH5α™ to ampicillin resistance. Plasmid DNAwas prepared and subjected to restriction enzyme analysis to identify aclone in which the cryET39 gene had inserted into pEG1915 in the properorientation. It was necessary for the sense strand of cryET39 to beoriented in the same direction as that of the cry2Ac regulatory regionfor efficient transcription to occur. Restriction digests using theenzymes shown in FIG. 2 identified a plasmid containing cryET39 in theproper orientation. This plasmid was designated pEG1921.

A Cry⁻ strain of B. thuringiensis was transformed to erythromycinresistance by the introduction of pEG1921. This recombinant strain,designated EG11937, was grown in C2 sporulation medium until sporulationand crystal formation had occurred. Phase contrast microscopy clearlyidentified crystalline inclusions in the shape of elongated rectangles,or needles, in the culture. The spores, crystals, and unlysed sporangiawere harvested by centrifugation. The material in the pellet was washedtwice in a solution of 0.005% Triton X-100®, 10 mM Tris-HCl pH7.5 andsuspended at one-half the original volume in the wash solution.

SDS-PAGE was used to visualize the protein in the crystal. 25 μl of 0.5N NaOH was added to 100 μl of the sample to inhibit proteolytic activitywhich can destroy the protein as the crystal is solubilized. After 2.5min at room temperature 65 μl of 3× Laemmli sample buffer (30% glycerol,15% 2-mercaptoethanol, 3% SDS, 0.1875 M Tris, 0.01% bromphenol blue) wasadded to the sample. The sample was heated to 100° C. for 5 min,centrifuged briefly to remove insoluble material, and loaded onto anacrylamide gel. The protein bands were visualized by staining withCoomassie Brilliant Blue R-250. This analysis demonstrated that EG11937expressed the 44-kDa CryET39 protein and not the 13-kDa (CryET74) or34-kDa (CryET75) proteins produced by recombinant strain, EG11529. ThePCR™-generated copy of the cryET39 gene in pEG1921 was sequenced toconfirm that it was identical to the wild-type copy from pEG1337. StrainEG11937 was grown and prepared for bioassays on WCRW larvae.Unexpectedly the crystal protein from EG11937 had no activity on theinsects. This result suggested that either the CryET39 protein requiresthe presence of the CryET74 to be toxic, or that CryET74 is the activetoxin protein.

pEG1337 was digested with the restriction enzymes HindIII and EcoRI torelease an approximately 3.2-kb fragment containing the cryET74 gene andonly a small piece of the cryET39 gene. This fragment was isolated on anagarose gel, purified, and cloned into the shuttle vector pHT315,digested with HindIII and EcoRI, using procedures described above. Thisplasmid, designated pEG1919, was introduced into the Cry⁻ B.thuringiensis strain, EG10650, by electroporation, transforming therecombinant cells to erythromycin resistance. One transformant,designated EG11935, was grown in C2 sporulation medium to determine ifthe cloned cryET74 gene could direct the expression of the crystalprotein. The culture was harvested and the crystal protein analyzed bySDS-PAGE as described above. EG11935 produced only CryET74 and had noactivity on larvae of the WCRW.

The observations that CryET39 and CryET74, individually, have noactivity on WCRW larvae indicates that the two proteins interact to forma toxic protein composition. PCR™ was used to generate a DNA fragmentcontaining the genes for CryET39 and CryET74, but not the gene forCryET75 also present on the 8.4-kb fragment of pEG1337 (see map ofpEG1337). The m13/pUC forward sequencing primer, (GibcoBRL), and mr45were used to amplify an approximately 3.7-kb product containing bothcryET74 and cryET39. PCR™ was performed using conditions described aboveusing pEG1337 as the template. The PCR™ product was gel purified,digested with HindIII, and cloned into pHT315 that had been cut withHindIII and treated with bacterial alkaline phosphatase. The resultingplasmid, designated pEG1920, was used to transform the Cry⁻ B.thuringiensis strain, EG10650, to erythromycin resistance. Onerecombinant, designated EG11936, was grown to assess crystal proteinproduction. EG11936 produced both the 44-kDa CryET39 and theapproximately 13-kDa CryET74 polypeptides. Crystal proteins produced byEG11936 had activity on WCRW larvae comparable to the activity seen withthe recombinant strain, EG11529.

5.12 Example 12 Toxicity of Crystal Proteins to Insects

5.12.1 Toxicity of EG11529 Crystal Proteins to SCRW Larvae

The toxicity to SCRW larvae (Diabrotica undecimpunctata howardi) wasdetermined for the recombinant strain EG11529, that expressed CryET39,CryET74, and CryET75 polypeptides.

EG11529 was grown in C2 medium at 30° C. for 3 days until sporulationand lysis had occurred. The cultures were harvested by centrifugation,washed twice in 1× original volume 0.005% Triton X-100®, and suspendedin 1/10 the original culture volume of 0.005% Triton X-100®. Forcomparison EG11535, a recombinant B. thuringiensis strain expressing thecoleopteran-toxic protein Cry3B2 (Donovan et al., 1992) was grown andharvested in the same manner. SDS-PAGE was used to visualize theproteins. The proteins were quantified by comparison with standardloading of a known amount of bovine serum albumin (Sigma Chemical Co.,St. Louis, Mo.) using a Computing Densitometer, Model 300A, (MolecularDynamics, Sunnyvale, Calif.), following the manufacturer's procedures.

SCRW larvae were bioassayed via surface contamination of an artificialdiet similar to Marrone et al., (1992), but without formalin. Eachbioassay consisted of eight serial aqueous dilutions with aliquotsapplied to the surface of the diet. After the diluent (an aqueous 0.005%Triton X-100® solution) had dried, first instar larvae were placed onthe diet and incubated at 28° C. Thirty-two larvae were tested per dose.Mortality was scored after 7 days. Data from replicated bioassays werepooled for probit analysis (Daum, 1970) with mortality being correctedfor control death, the control being diluent only (Abbot, 1925). Resultswere reported as the amount of crystal protein per well (175 mm² of dietsurface) resulting in an LC₅₀, the concentration killing 50% of the testinsects. 95% confidence intervals were also reported.

TABLE 14 INSECTICIDAL ACTIVITY OF EG11529 PROTEINS ON SCRW LARVAE LC₅₀Sample (μg protein/well) 95% C.I. EG11529 34.1 28-41 EG11535 (Cry3B2)49.5 33-83

The results shown in the above table demonstrated that the crystalproteins of EG11529 had significant activity on larvae of the SCRW. TheLC₅₀ value for EG11529was lower than that seen for the Cry3B2 controlprotein, although the 95% confidence intervals did overlap, indicatingthe difference may not have been significant.

5.12.2 Toxicity of CryET39 and CryET74 to WCRW Larvae

The toxicity to WCRW larvae (Diabrotica virgifera virgifera) wasdetermined for EG11529, as well as the recombinant strains constructedto produce the individual crystal proteins of EG11529. The recombinantstrains and the crystal proteins they produced are shown in Table 15.

TABLE 15 Bt Recombinant Strain Crystal Protein Expressed MW (kDa)*EG11529 CryET39 44 kDa CryET74 13 kDa CryET75 34 kDa EG11934 CryET75 34kDa EG11935 CryET74 13 kDa EG11936 CryET39 + CryET74 44 kDa + 13 kDaEG11937 CryET39 44 kDa *Molecular weights are estimated by migration ofthe protein on an SDS-PAGE gel and comparison with standards of knownmolecular weight.

A series of bioassays to determine the activity of the crystal proteinswas performed essentially as described for the SCRW assays, with theexception that neonate larvae were used instead of first instar larvae.Purified crystal proteins were prepared for the first assay usingsucrose step gradients. EG11529 was grown for three days at 30° C. in C2sporulation medium. The sporulated and lysed cultures were harvested bycentrifugation and washed, twice, in equal volumes of wash buffer (10 mMTris-HCl, pH 7.5, 0.005% Triton X-100®), and suspended at 1/10^(th) theoriginal volume in the wash solution. Sucrose step gradients wereprepared by layering solutions of decreasing concentrations of sucrose,in the wash solution, in 25×89 mm Ultra-Clear centrifuge tubes (BeckmanInstruments, Inc., Palo Alto, Calif.). The steps consisted of 7.5 mleach of the following concentrations of sucrose (bottom to top):79%-72%-68%-55%. 5 ml of the spore/crystal suspension were layered ontop of the gradient. The gradients were centrifuged at 18,000 rpm at 4°C. in an L8-70M ultracentrifuge (Beckman Instruments) overnight. Thecrystal proteins of EG11529 separated into two distinct bands. One band,at the 68%-72% interface, contained only the CryET75 protein. The secondband, at the 72%-79% interface, contained both CryET39 and CryET74. Thebands were pulled off with a pipet and washed, twice, in the washbuffer. The protein sample was then run over a second gradient to assurea complete separation of CryET75 from CryET39 and CryET74. The proteinsamples were run on an SDS-PAGE gel to verify the sample integrity. Thesamples were then quantified using a standard protein assay (Bio-RadLaboratories, Hercules, Calif.), following manufacturer's procedures.

An assay was performed comparing the toxicity to WCRW larvae of theCryET39+CryET74 and the CryET75 purified crystal protein samples withthe toxicity of EG11529. EG11529 was prepared as a spore/crystalsuspension and the amount of protein was determined by SDS-PAGE anddensitometry. Data from the assay were pooled for probit analysis (Daum,1970) with mortality being corrected for control death, the controlbeing diluent only (Abbot, 1925). Results are reported as the amount ofcrystal protein per well (175 mm² of diet surface) resulting in an LC₅₀,the concentration killing 50% of the test insects. 95% confidenceintervals were also reported in Table 16.

TABLE 16 INSECTICIDAL ACTIVITY OF EG11529 PROTEINS ON WCRW LARVAE SampleLC₅₀ (μg/well) 95% C.I. CryET75 No Activity* EG11529 8.6 6.6-10.6CryET39 + CryET74 9.7 7.2-12.7 *6% mortality at a dose of 45 μg/well

This assay clearly demonstrated that the purified CryET75 protein wasnot toxic towards the larvae of the WCRW. The sample containing themixture of CryET39 and CryET74 had activity similar to that of EG11529,indicating that the CryET75 played no synergistic role in the toxicityof EG11529 to WCRW larvae.

To determine if the CryET74 is the toxic component of the EG11529 straina spore/crystal suspension of EG11935, which produces only CryET74, wascompared in bioassay to spore/crystal suspensions of EG11529 andEG11936, which produces both CryET39 and CryET74. Data from replicatedbioassays were pooled for probit analysis (Daum, 1970) with mortalitybeing corrected for control death, the control being diluent only(Abbot, 1925). Results are reported as the amount of crystal protein perwell (175 mm² of diet surface) resulting in an LC₅₀, the concentrationkilling 50% of the test insects. 95% confidence intervals are alsoreported in Table 17 below.

TABLE 17 INSECTICIDAL ACTIVITY OF B. thuringiensis PROTEINS ON WCRWLARVAE Sample LC₅₀ (μg/well) 95% C.I. EG11935 No Activity at 80 μg/wellEG11529  9.78 6.9-12.5 EG11936 14.5 9.7-19.5

The CryET74 protein produced by EG11935 had no activity on WCRW larvae,suggesting that the CryET39 protein, either alone or in combination withCryET74, was responsible for the insecticidal activity seen in EG11529and EG11936.

An assay comparing a spore/crystal suspension of EG11937, which producesonly the CryET39 crystal protein, with suspensions of EG11936 andEG11937 was performed. Also included in this assay were 50:50 mixturesof EG11935+EG11937 to see if a mixture of CryET39 and CryET74 hadactivity similar to that of EG11936. The data (Table 18) are expressedas percent control, which is mortality at a given dose corrected forcontrol mortality in the diluent control. Two identical samples ofEG11937 were prepared for the purposes of repetition.

TABLE 18 Sample Dose (μg/well) Percent Control EG11935 80 0 EG11935 1600 EG11936 80 100 EG11936 160 100 EG11937 (1) 80 10.5 EG11937 (1) 160 6.7EG11937 (2) 80 13.3 EG11937 (2) 160 0 EG11935 + EG11937 (1) 80 100EG11935 + EG11937 (1) 160 100 EG11935 + EG11937 (2) 80 100 EG11935 +EG11937 (2) 160 93.3

The results of this assay clearly demonstrated that CryET39 proteinalone, as expressed in EG11937, does not account for the activity seenin EG11936 or EG11529. The addition of CryET74 to the CryET39 protein,however, resulted in a composition toxic to larvae of the WCRW. Thesedata suggest that CryET39 and CryET74 interact to form the toxiccomponent of EG11529 and EG11936.

5.12.3 Toxicity of the Crystal Proteins of EG11529 to CPB Larvae

A sporulated culture of EG11529 was harvested, washed and suspended asdescribed above, to determine if the crystal proteins produced byEG11529 are toxic to the larvae of the Colorado potato beetle (CPB). Theassay on CPB larvae was performed using techniques similar to those inthe SCRW assay, except for the substitution of BioServe's #9380 insectdiet (with potato flakes added) for the artificial diet. Mortality wasscored at three days instead of seven days. For this assay 16 insectswere used at a single dose of 140 μg/well. At this dose 100% of thelarvae were killed demonstrating that EG11529 is toxic to CPB larvae.

5.13 Example 13 Identification of Genes Encoding Related δ-EndotoxinPolypeptides

B. thuringiensis strains producing crystal proteins of 40-50 kDa wereidentified by SDS-PAGE analysis of parasporal crystals produced bysporulating cultures. Total DNA was extracted from these strainsfollowing procedures described above, digested with the restrictionendonuclease HindIII, and the restriction fragments resolved by agarosegel electrophoresis and blotted to nitrocellulose filters for Southernblot analysis. PCR™ was used to amplify a segment of the cryET39 genefor use as a hybridization probe to identify and clone related toxingenes from these B. thuringiensis strains. The PCR™ fragment extendedfrom nucleotide 176 of the cryET39 coding sequence to approximately200-bp 3′ to the end of the gene and was generated using the opposingprimers mr13 and mr24 and plasmid pEG1337 as a template.

mr13: 5′-TGACACAGCTATGGAGC-3′ (SEQ ID NO:33) mr24:5′-ATGATTGCCGGAATAGAAGC-3′ (SEQ ID NO:34)

The amplified DNA fragment was radioactively labeled using α-³²P-dATPand a random primer labeling kit (Prime-a-Gene® Labeling System; PromegaCorporation, Madison, Wis.). Following incubation with thecryET39-specific hybridization probe, the filters were washed undermoderately stringent conditions (e.g., in 0.1×-1.0×SSC at 55 C), andexposed to X-ray film to obtain an autoradiogram identifying DNAfragments containing cryET39-related sequences. Several strains yieldedhybridization patterns that differed from that of EG5899. Three strains,designated EG4100, EG4851, and EG9444 respectively, were selected forfurther characterization.

The cloning and expression of the cry genes from strains EG4100, EG4851,and EG9444 was accomplished using procedures described in Section 5.4,Section 5.5and Section 5.6. DNA was prepared from the strains andpartially digested with the restriction enzyme MboI, resulting in anassortment of essentially random DNA fragments. The MboI fragments wereresolved on an agarose gel and fragments in the 6-10-kb size range werepurified. The purified MboI fragments were then ligated into a B.thuringiensis/E. coli shuttle vector, pHT315, previously digested withBamHI and treated with alkaline phosphatase. The ligation mixure wasthen used to transform E. coli to ampicillin resistance, thusconstructing a library of cloned fragments representing the genome ofeach respective B. thuringiensis strain. The E. coli libraries wereplated on LB agar containing 50 ug/ml ampicillin and the coloniestransferred to nitrocellulose filters. To identify cryET39-relatedsequences the filters were probed with either the radiolabeledoligonucleotide wd271 (EG9444 library), as described in Section 5.4 andSection 5.5, or with the cryET39-specific hybridization probe describedabove (EG4100 and EG4851 libraries). Plasmid DNA was isolated fromhybridizing E. coli colonies and used to transform an acrystalliferousB. thuringiensis host strain to erythromycin resistance. Recombinant B.thuringiensis clones were grown to sporulation in C2 medium and crystalproteins were analyzed by SDS-PAGE as described in Section 5.6.

A cloned fragment identified in the manner described above from theEG4100 library encoded an approximately 60-kDa crystal protein,designated CryET69 (SEQ ID NO:14). DNA sequence analysis revealed thatthe cryET69 gene (SEQ ID NO:13) encoded a protein of 520 amino acidresidues. The CryET69 protein showed ˜23% sequence identity to CryET39.The recombinant B. thuringiensis strain expressing CryET69 wasdesignated EG11647 and the recombinant plasmid containing the cryET69gene was designated pEG1820. EG4100 and EG11647 were deposited with theARS Patent Culture Collection and given the NRRL accession numbersB-21786 and B-21787, respectively.

A cloned fragment isolated from the EG9444 library as described aboveencoded an approximately 45-kDa crystal protein, designated CryET71,that was related to CryET39, and an approximately 14-kDa crystalprotein, designated CryET79, that was related to CryET74. DNA sequenceanalysis revealed that the cryET71 gene (SEQ ID NO:11) encodes a proteinof 397 amino acids and that the cryET79 gene (SEQ ID NO:9) encodes aprotein of 123 amino acids. The CryET71 protein (SEQ ID NO:12) showed78% sequence identity to CryET39 while the CryET79 protein (SEQ IDNO:10) showed 80% sequence identity to CryET74. The recombinant B.thuringiensis strain expressing CryET71 and CryET79 was designatedEG11648 and the recombinant plasmid containing the cryET71 and cryET79genes was designated pEG1821 (Table 9). EG11648 was toxic to larvae ofthe WCRW. By analogy to the related CryET39 and CryET74 proteins, it waspresumed that both CryET71 and CryET79 were required for full WCRWtoxicity. EG9444 and EG11648 have been deposited with the ARS PatentCulture Collection and given the NRRL accession numbers B-21785 andB-21788, respectively.

A cloned fragment isolated from the EG4851 library as described aboveencoded an approximately 44-kDa crystal protein, designated CryET76,that was related to CryET39, and an approximately 15-kDa protein,designated CryET80, that was related to CryET74. DNA sequence analysisrevealed that the cryET76 gene (SEQ ID NO:1) encoded a protein of 387amino acids and that the cryET80 gene (SEQ ID NO:3) encoded a protein of132 amino acids. The CryET76 protein (SEQ ID NO:2) showed 61% sequenceidentity to CryET39 while the CryET80 protein SEQ ID NO:4) showed 52%sequence identity to CryET74. The recombinant B. thuringiensis strainexpressing CryET76 and CryET80 was designated EG11658, and therecombinant plasmid containing the cryET76 and cryET80 genes has beendesignated pEG1823 (Table 9). EG11658 was toxic to larvae of the WCRW.By analogy to the related CryET39 and CryET74 proteins, it was presumedthat both CryET76 and CryET80 were required for full WCRW toxicity.EG4851 and EG11658 were deposited with the ARS Patent Culture Collectionand given the NRRL accession numbers B-21915 and B-21916, respectively.

Based on these results, the inventors contemplate that the utilizationof procedures similar to those described herein will lead to thediscovery and isolation of additional B. thuringiensis crystal proteintoxins. DNA probes, based on the novel sequences disclosed herein may beprepared from oligonucleotides, PCR™ products, or restriction fragmentsand used to identify additional genes related to those described herein.These new genes may also be cloned, characterized by DNA sequencing, andtheir encoded proteins evaluated in bioassay on a variety of insectpests using the methods described herein. Novel genes, in turn, maytherefore result in the identification of new families of related genes,as seen in the above Examples.

5.14 Example 14 Sequencing of Related Cry Genes

5.14.1 CryET71

An initial nucleotide sequence for the cryET71 gene was obtained usingthe oligonucleotide wd271 as a sequencing primer and proceduresdescribed in Section 5.7. Successive oligonucleotides to be used forpriming sequencing reactions were designed from the sequencing data ofthe previous set of reactions to obtain the complete the sequence of thecryET71 gene.

The DNA sequence of the CryET71 gene is represented by SEQ ID NO:11, andencodes the amino acid sequence of the CryET71 polypeptide, representedby SEQ ID NO:12.

5.14.1.4 Characteristics of the CryET71 Polypeptide

The CryET71 polypeptide comprises a 397-amino acid sequence, has acalculated molecular mass of 45,576 Da, and has a calculated pI equal to4.75. The amino acid composition of the CryET71 polypeptide is given inTable 19.

TABLE 19 AMINO ACID COMPOSITION OF CRYET71 Amino Acid # Residues % TotalAla 11 2.7 Arg 8 2.0 Asn 38 9.5 Asp 28 7.0 Cys 2 0.5 Gln 25 6.2 Glu 225.5 Gly 19 4.7 His 5 1.2 Ile 41 10.3 Leu 31 7.8 Lys 30 7.5 Met 8 2.0 Phe6 1.5 Pro 16 4.0 Ser 29 7.3 Thr 33 8.3 Trp 7 1.7 Tyr 24 6.0 Val 14 3.5Acidic (Asp + Glu) 50 Basic (Arg + Lys) 38 Aromatic (Phe + Trp + Tyr) 37Hydrophobic (Aromatic + Ile + Leu + Met + Val) 1315.14.2 CryET79

An initial sequence for the upstream cryET79 gene was obtained using anoligonucleotide primer designed from the completed cryET71 sequence. DNAsamples were sequenced using the ABI PRISM™ DyeDeoxy sequencingchemistry kit (Applied Biosystems) according to the manufacturer'sprotocol. The completed reactions were run on as ABI 377 automated DNAsequencer. DNA sequence data were analyzed using Sequencher v3.0 DNAanalysis software (Gene Codes Corp.). Successive oligonucleotides to beused for priming sequencing reactions were designed from the sequencingdata of the previous set of reactions to obtain the complete cryET79gene sequence.

5.14.2.3 Characteristics of the CryET79 Polypeptide

The CryET79 polypeptide comprises a 123-amino acid sequence, has acalculated molecular mass of 13,609 Da, and has a calculated pI equal to6.32. The amino acid composition of the CryET79 polypeptide is given inTable 20.

TABLE 20 AMINO ACID COMPOSITION OF CRYET79 Amino Acid # Residues % TotalAla 5 4.0 Arg 4 3.2 Asn 12 9.7 Asp 5 4.0 Cys 0 0 Gln 6 4.8 Glu 7 5.6 Gly13 10.5 His 6 4.8 Ile 6 4.8 Leu 4 3.2 Lys 6 4.8 Met 2 1.6 Phe 3 2.4 Pro3 2.4 Ser 13 10.5 Thr 13 10.5 Trp 1 0.8 Tyr 8 6.5 Val 6 4.8 Acidic(Asp + Glu) 12 Basic (Arg + Lys) 10 Aromatic (Phe + Trp + Tyr) 12Hydrophobic (Aromatic + Ile + Leu + Met + Val) 305.14.3 CryET69

The NH₂-terminal amino acid sequence of the isolated CryET69 protein wasdetermined using procedures described in Section 5.3. The NH₂-terminalsequence of the isolated protein was:

(SEQ ID NO:29)  1   2   3   4   5   6   7   8   9  10  11 Met Asn ValAsn His Gly Met Ser Cys Gly Cys

An oligonucleotide primer based on the NH₂-terminal amino acid sequenceof the CryET69 protein was designed for use in sequencing cryET69. Thisoligonucleotide, designated crc12, has the following sequence:

5′-ATGAATGTAAATCATGGGATGWSNTGT-3′ (SEQ ID NO:30)where W=A and T and S=C and G. An initial nucleotide sequence wasobtained using crc12 as a sequencing primer and procedures described inSection 5.7. Successive oligonucleotides to be used for primingsequencing reactions were designed from the sequencing data of theprevious set of reactions. The completion of the sequence was achievedusing automated sequencing. DNA samples were sequenced using the ABIPRISM DyeDeoxy sequencing chemistry kit (Applied Biosystems) accordingto the manufacturer's protocol. The completed reactions were run on asABI 377 automated DNA sequencer. DNA sequence data were analyzed usingSequencher v3.0 DNA analysis software (Gene Codes Corp.).5.14.3.3 Characteristics of the CryET69 Polypeptide

The CryET69 polypeptide comprises a 520-amino acid sequence, has acalculated molecular mass of 58,609 Da, and has a calculated pI equal to5.84. The amino acid composition of the CryET69 polypeptide is given inTable 21.

TABLE 21 AMINO ACID COMPOSITION OF CRYET69 Amino Acid # Residues % TotalAla 24 4.6 Arg 30 5.7 Asn 60 11.5 Asp 27 5.1 Cys 9 1.7 Gln 32 6.1 Glu 244.6 Gly 32 6.1 His 9 1.7 Ile 24 4.6 Leu 31 5.9 Lys 15 2.8 Met 10 1.9 Phe20 3.8 Pro 24 4.6 Ser 39 7.5 Thr 48 9.2 Trp 6 1.1 Tyr 22 4.2 Val 34 6.5Acidic (Asp + Glu) 51 Basic (Arg + Lys) 45 Aromatic (Phe + Trp + Tyr) 48Hydrophobic (Aromatic + Ile + Leu + Met + Val) 147

5.16 Example 16 Database Searches for CryET69-Related Proteins

The deduced amino acid sequence for CryET69 was used to query theSWISS-PROT ALL and nr databases using FASTA and BLASTP as described forCryET39 in Section 5.8 except that the blosum50 comparison matrix wasused for the FASTA search. The results of the FASTA search indicatedthat CryET69 showed ˜32% sequence identity over a 338-amino acid regionwith the 42-kDa mosquitocidal crystal protein of B. sphaericus and ˜30%sequence identity over a 440-amino acid region with the 51-kDa crystalprotein of B. sphaericus.

5.17 Example 17 Database Searches for CryET71- and CryET79-RelatedProteins

The deduced amino acid sequences for CryET71 and CryET79 were used toquery the SWISS-PROT ALL and nr databases using FASTA and BLASTP asdescribed for CryET39 in Section 5.8. The results of the FASTA searchindicated that CryET71 showed ˜25% sequence identity over a 323-aminoacid region with the 42-kDa mosquitocidal crystal protein of B.sphaericus and ˜25% sequence identity over a 388-amino acid region withthe 51-kDa crystal protein of B. sphaericus. The FASTA and BLASTPsearches did not identify proteins with significant sequence identity toCryET79.

5.18 Example 18 Sequencing of the CryET76 and CryET80 Genes

A partial DNA sequence of the genes cloned on pEG1823 was determinedfollowing established dideoxy chain-termination DNA sequencingprocedures (Sanger et al., 1977). Preparation of the double strandedplasmid template DNA was accomplished using a Wizard® SV Miniprep Kit(Promega Corp.) following the manufacturer's procedures or a QiagenPlasmid Kit (Qiagen Inc.) following the manufacturer's procedures,followed by a phenol:chloroform:isoamyl alcohol (50:48:2) extraction andthen a chlorform:isoamyl alcohol (24:1) extraction. The sequencingreactions were performed using the Sequenase™ Version 2.0 DNA SequencingKit (United States Biochemical/Amersham Life Science Inc.) following themanufacturer's procedures and using ³⁵S-[dATP] as the labeling isotope(DuPont NEN® Research Products). Denaturing gel electrophoresis of thereactions was performed on a 6% (wt./vol.) acrylamide, 42% (wt./vol.)urea sequencing gel or on a CastAway™ Precast 6% acrylamide sequencinggel (Stratagene). The dried gel was exposed to Kodak X-OMAT AR X-rayfilm (Eastman Kodak) overnight at room temperature to obtain anautoradiogram.

A partial DNA sequence for the cryET76 and cryET80 genes on pEG1823 wasobtained by using the procedures described above. The cryET39-specificoligonucleotide mr18 was used as the initial sequencing primer. Thesequence of mr18 is:

5′-GTACCAGAAGTAGGAGG-3′ (SEQ ID NO:31)

Successive oligonucleotides to be used for priming sequencing reactionswere designed from the sequencing data of the previous set of reactions.The completion of the sequence was achieved using automated sequencing.DNA samples were sequenced using the ABI PRISM DyeDeoxy sequencingchemistry kit (Applied Biosystems) according to the manufacturer'ssuggested protocol. The completed reactions were run on as ABI 377automated DNA sequencer. DNA sequence data were analyzed usingSequencher v3.0 DNA analysis software (Gene Codes Corp.). The DNAsequence of cryET76 (SEQ ID NO:1) and cryET80 (SEQ ID NO:3) is shownbelow. The deduced amino acid sequence of the CryET76 protein (SEQ IDNO:2) and the CryET80 protein (SEQ ID NO:4) is also shown below. Theentire sequenced region is shown in (SEQ ID NO:17).

5.18.1 CryET76

The DNA sequence of the CryET76 gene is represented by SEQ ID NO:1, andencodes the amino acid sequence of the CryET76 polypeptide, representedby SEQ ID NO:2.

5.18.1.3 Characteristics of the CryET76 Polypeptide

The CryET76 polypeptide comprises a 387-amino acid sequence, has acalculated molecular mass of 43,812 Da, and has a calculated pI equal to5.39. The amino acid composition of the CryET76 polypeptide is given inTable 22.

TABLE 22 AMINO ACID COMPOSITION OF CRYET76 Amino Acid # Residues % TotalAla 14 3.6 Arg 7 1.8 Asn 39 10.0 Asp 17 4.3 Cys 1 0.2 Gln 17 4.3 Glu 225.6 Gly 22 5.6 His 4 1.0 Ile 31 8.0 Leu 34 8.7 Lys 27 6.9 Met 5 1.2 Phe8 2.0 Pro 10 2.5 Ser 30 7.7 Thr 47 12.1 Trp 8 2.0 Tyr 24 6.2 Val 20 5.1Acidic (Asp + Glu) 39 Basic (Arg + Lys) 34 Aromatic (Phe + Trp + Tyr) 40Hydrophobic (Aromatic + Ile + Leu + Met + Val) 1305.18.2 CryET80

The DNA sequence of the CryET80 gene is represented by SEQ ID NO:3, andencodes the amino acid sequence of the CryET80 polypeptide, representedby SEQ ID NO:4.

5.18.2.3 Characteristics of the CryET80 Polypeptide

The CryET80 polypeptide comprises a 132-amino acid sequence, has acalculated molecular mass of 14,839 Da, and has a calculated pI equal to6.03. The amino acid composition of the CryET80 polypeptide is given inTable 23.

TABLE 23 AMINO ACID COMPOSITION OF CRYET80 Amino Acid # Residues % TotalAla 7 5.3 Arg 8 6.0 Asn 13 9.8 Asp 8 6.0 Cys 1 0.7 Gln 3 2.2 Glu 8 6.0Gly 9 6.8 His 8 6.0 Ile 13 9.8 Leu 6 4.5 Lys 4 3.0 Met 2 1.5 Phe 2 1.5Pro 3 2.2 Ser 11 8.3 Thr 11 8.3 Trp 1 0.7 Tyr 6 4.5 Val 8 6.0 Acidic(Asp + Glu) 16 Basic (Arg + Lys) 12 Aromatic (Phe + Trp + Tyr) 9Hydrophobic (Aromatic + Ile + Leu + Met + Val) 385.18.3 Characteristics of the CryET76, CryET80 and CryET84 GenesIsolated from EG4851 (SEQ ID NO:17)

The DNA sequence of the entire three gene operon containing the CryET76,CryET80, and CryET84 coding regions is represented by SEQ ID NO:17.

In strain EG4851, the cryET84 gene is located immediately 5′ to thecryET80 and cryET76 genes. The cryET84 gene begins at nucleotide 656 andends at nucleotide 1681. The cryET80 gene begins at nucleotide 1773 andends at nucleotide 2168. The cryET76 gene begins at nucleotide 2264 andends at nucleotide 3424.

5.19 Example 19 Analysis of Sequence Homologies

5.19.1 Database Searches for CryET76- and CryET80-Related Proteins

The amino acid sequences of the CryET76 and CryET80 proteins, deduced bytranslation of the nucleotide sequence, were used to query sequencedatabases for related protein sequences. The SWISS-PROT ALL database wasqueried using FASTA version 3.15 (Pearson and Lipman, 1988) provided bythe FASTA server at European Biotechnology Institute using the followingparameters (library=swall, matrix=pam150, ktup=2, gapcost=−12,gapxcost=−2). The amino acid sequences of CryET76 and CryET80 were alsoused to query the non-redundant (nr) database of the National CenterBiotechnology Information (NCBI) using BLASTP version 2.0 (Altschul etal., 1997) with the following parameters: matrix=blosum 62, gappedalignment, other parameters=default settings.

The results of the FASTA analysis revealed that CryET76 showed ˜27%sequence identity over a 320-amino acid region with the 42-kDamosquitocidal crystal protein from B. sphaericus while CryET80 showed nosignificant sequence similarity to sequences in SWISS-PROT ALL. Theresults of the BLASTP search were in general agreement with those of theFASTA search. No proteins with significant sequence similarity toCryET80 were identified.

5.19.2 Additional Sequence Comparisons with Cry Proteins

Sequence alignments were performed to compare the CryET39, CryET74,CryET75, CryET71, CryET79, CryET76, CryET80 and CryET69 sequences withsequences from recently published patent applications. The alignmentswere performed using PALIGN in the PC/GENE version 6.85 sequenceanalysis package (Intelligenetics Corp. Mountain View, Calif.). Thepairwise alignments were performed using the following parameters:comparison matrix=unitary, open gap cost=3, unit gap cost=1.

5.19.2.1 CryET39

Sequence alignment comparing the CryET39 sequence with sequences fromrecently published patent applications revealed sequence similaritybetween CryET39 and proteins identified by sequence identifiers 11, 38and 43 of Intl. Pat. Appl. Publ. No. WO 97/40162. CryET39 showed a 99.2%sequence identity to sequence number 11, 78.6% sequence identity tosequence number 38, and 79.9% sequence identity to sequence number 43.

5.19.2.2 CryET74

Sequence alignment comparing the CryET74 sequence with sequences fromrecently published patent applications revealed sequence similaritybetween CryET74 and sequence identifier numbers 32, 36, and 41 of theIntl. Pat. Appl. Publ. No. WO 97/40162. CryET74 shows 100% sequenceidentity with sequence identifier number 32, 80.7% sequence identitywith sequence identifier number 36, and 77.3% sequence identity withsequence identifier number 41 of that application.

5.19.2.3 CryET75

Sequence alignment comparing CryET75 with sequences from recentlypublished patent applications revealed approximately 26% sequenceidentity between CryET75 and CryET33, a coleopteran-toxic protein,disclosed in Intl. Pat. Appl. Publ. No. WO 97/17600.

5.19.2.4 CryET71

Sequence alignment comparing the CryET71 sequence with sequences fromrecently published patent applications revealed sequence similaritybetween CryET71 and sequence identifier numbers 11, 38, and 43 of theIntl. Pat. Appl. Publ. No. WO 97/40162. CryET71 showed 78.4% sequenceidentity with sequence identifier number 11; 91.9% sequence identitywith sequence identifier number 38; and 97.4% sequence identity withsequence identifier number 43.

5.19.2.5 CryET79

Sequence alignment comparing the CryET79 sequence with sequences fromrecently published patent applications revealed sequence similaritybetween CryET79 and the sequences identifier numbers 32, 36, and 41 ofthe Intl. Pat. Appl. Publ. No. WO 97/40162. CryET79 showed 79.8%sequence identity with sequence identifier number 32; 95.9% sequenceidentity with sequence identifier number 36; and 91% sequence identitywith sequence identifier number 41.

5.19.2.6 CryET76

Sequence alignment comparing the CryET76 sequence with sequences fromrecently published patent applications revealed sequence similaritybetween CryET76 and sequence identifier numbers 11, 38, and 43 of theIntl. Pat. Appl. Publ. No. WO 97/40162. CryET76 showed 60.8% sequenceidentity to sequence identifier number 11; 61.6% sequence identity tosequence identifier number 38; and 61.9% sequence identity to sequenceidentifier number 43.

5.19.2.7 CryET80

Sequence alignment comparing the CryET80 sequence with sequences fromrecently published patent applications revealed sequence similaritybetween CryET80 and sequence identifier numbers 32, 36, and 41 of theIntl. Pat. Appl. Publ. No. WO 97/40162. CryET80 showed 52.1% sequenceidentity to sequence identifier number 32; 56.1% sequence identity tosequence identifier number 36; and 54.5% sequence identity to sequenceidentifier number 41.

5.19.2.8 CryET69

Sequence alignment comparing the CryET69 sequence revealed only 23-25%sequence identity between CryET69 and sequence identifier numbers 11,38, and 43 of the Intl. Pat. Appl. Publ. No. WO 97/40162. This crystalprotein showed a higher degree of homology to the mosquitocidal crystalproteins of B. sphaericus than to the crystal proteins of B.thuringiensis.

5.19.3 Summary

These analyses demonstrated that the amino acid sequences of CryET69,CryET75, CryET76 and CryET80 are markedly different from the sequencesof previously described insecticidal crystal proteins. Employing thenomenclature established for B. thuringiensis crystal proteins(Crickmore et al., 1998), CryET76 and CryET80 would be assigned a newsecondary rank and CryET69 and CryET75 would be assigned a new primaryrank.

5.20 Example 20 Expression of Recombinant CryET76 and CryET80Polypeptides

To characterize the properties of the CryET76 and CryET80 proteins, itwas necessary to express the cloned cryET76 and cryET80 genes in a B.thuringiensis strain that did not produce other crystal proteins (i.e. aCry⁻ strain). The plasmid containing the cloned cryET76 and cryET80genes, pEG1823, contains a B. thuringiensis origin of replication aswell as an origin that directs replication in E. coli, as describedabove. pEG1823 was used to transform the Cry⁻ B. thuringiensis strainEG10650 to erythromycin resistance (Em^(R)) by electroporation (Macalusoand Mettus, 1991). Cells transformed to Em^(R) were selected byincubation overnight on LB agar plates containing 25 μg/ml erythromycin.One Em^(R) colony from each transformation was selected for furtheranalysis. One isolate was designated EG11658.

EG11658 was grown in C2 sporulation medium containing 25 μg/mlerythromycin for four days at 25° C., at which point sporulation andcell lysis had occurred. Microscopic examination of the sporulatedcultures demonstrated that the recombinant strain was producingparasporal inclusions. The sporulated culture of EG11658 was harvestedby centrifugation, washed, and resuspended at one-tenth the originalvolume in H₂O. The crystal protein in the suspension was characterizedby SDS-PAGE analysis which revealed the production of approximately 44-and 15-kDa proteins.

5.21 Example 21 Toxicity of CryET76 and CryET80 to Insects

The toxicity of CryET76 and CryET80 protein towards WCRW was determined.

EG11658 was grown in C2 medium at 25° C. for four days until sporulationand cell lysis had occurred. The culture was harvested bycentrifugation, washed in approximately 2.5 times the original volumewith distilled H₂O, and resuspended in 0.005% Triton X-100® at one-tenththe original volume. For comparison with EG11658, the recombinant B.thuringiensis strains, EG11529, producing the WCRW-toxic proteinsCryET39 and CryET74, and EG11648, producing the WCRW-toxic proteinsCryET71 and CryET79, were grown and harvested in the same manner. Toxinproteins from the samples were quantified by SDS-PAGE as described(Brussock and Currier, 1990. The procedure was modified to eliminate theneutralization step with 3M HEPES.

WCRW larvae were bioassayed via surface contamination of an artificialdiet (20 g agar, 50 g wheatgerm, 39 g sucrose, 32 g casein, 14 g fiber,9 g Wesson salts mix, 1 g methyl paraben, 0.5 g sorbic acid, 0.06 gcholesterol, 9 g Vanderzant's vitamin mix, 0.5 ml linseed oil, 2.5 mlphosphoric/propionic acid per 1 liter). Each bioassay of EG11658(CryET76 and CryET80), EG11529 (CryET39 and CryET74), and EG11648(CryET71 and CryET79) consisted of eight serial aqueous dilutions withaliquots applied to the surface of the diet. After the diluent (anaqueous 0.005% Triton X-100® solution) had dried, neonate larvae wereplaced on the diet and incubated at 28° C. Thirty-two larvae were testedper dose. Mortality was scored after seven days. Data from replicatedbioassays were pooled for probit analysis (Daum, 1970) with mortalitybeing corrected for control death, the control being diluent only(Abbott, 1925). Results are reported as the amount of crystal proteinper well (175 mm² of diet surface) resulting in an LC₅₀, theconcentration killing 50% of the test insects. 95% confidence intervalsare also reported for the LC₅₀ values (Table 24).

TABLE 24 INSECTICIDAL ACTIVITY OF CRY PROTEINS TO WCRW LARVAE CrystalLC₅₀ LC₉₅ Sample Protein (μg protein/well) 95% C.I. (μg protein/well)EG11658 CryET76 10.7  2.2-18.9 46 CryET80 EG11648 CryET71 5.3  0.9-10.127 CryET79 EG11936 CryET39 12.3 12.5-14.3 32 CryET74

The results shown in Table 24 demonstrated that the CryET76 and CryET80proteins had significant activity on WCRW larvae.

5.22 Example 22 Toxicity of CryET69 to Insects

The toxicity of CryET69 towards WCRW was determined using proceduresdescribed in Section 5.21. Results are reported as the amount of crystalprotein per well (175 mm² of diet surface) resulting in an LC₅₀, theconcentration killing 50% of the test insects. 95% confidence intervalsare also reported for the LC₅₀ values (Table 25).

TABLE 25 INSECTICIDAL ACTIVITY OF CRYET69 TO WCRW LARVAE Crystal LC₅₀LC₉₅ Sample Protein (μg protein/well) 95% C.I. (μg protein/well) EG11204Cry3B2 13.8 3.2-30.1 502 EG11647 CryET69 147.3  73-1292 6190 Controlmortality = 22%These results demonstrated that CryET69 was significantly less activethan Cry3B2 against WCRW. Nevertheless, this crystal protein apparentlyrepresents a new class of coleopteran-toxic δ-endotoxins.

5.23 Example 23 Construction of Strains EG12156 and EG12158

Recombinant B. thuringiensis strains were constructed that produceeither CryET76 or CryET80. A frameshift mutation was introduced into thecryET76 coding sequence on pEG1823 to generate a recombinant plasmidcapable of directing the production of CryET80 alone. A unique AgeIrestriction site within the cryET76 coding sequence was identified bycomputer analysis of the determined cryET76 nucleotide sequence.Subsequent digestion of pEG1823 with AgeI confirmed that thisrestriction site was unique to the plasmid. To generate a frameshiftmutation at this site, pEG1823 was digested with AgeI and the DNA endsblunt-ended with T4 polymerase in the presence of dNTPs. The linear DNAfragment was subsequently resolved by electrophoresis on a 1% agarosegel, the DNA band excised with a razor blade, and the DNA purified usingthe Qiagen gel extraction kit. The purified DNA was self-ligated usingT4 ligase and used to transform the E. coli strain DH5α to ampicillinresistance. Restriction enzyme analysis of DNA recovered from severalampicillin-resistant clones confirmed the disruption of the AgeI site onpEG1823. The recombinant plasmid from one such clone was designatedpEG2206. pEG2206 was subsequently used to transform, viaelectroporation, the acrystalliferous B. thuringiensis strain EG10650 toerythromycin resistance. The recombinant Bt strain containing pEG2206was designated EG12156.

A deletion mutation was introduced into the cryET80 coding sequence onpEG1823 to generate a recombinant plasmid capable of directing theproduction of CryET76 alone. A unique DraIII restriction site within thecryET80 coding sequence was identified by computer analysis of thedetermined cryET80 nucleotide sequence. Subsequent digestion of pEG1823with DraIII confirmed that this restriction site was unique to theplasmid. To generate a mutation at this site, pEG1823 was digested withDraIII and the DNA ends blunt-ended with T4 polymerase in the presenceof dNTPs. The linear DNA fragment was subsequently resolved byelectrophoresis on a 1% agarose gel, the DNA band excised with a razorblade, and the DNA purified using the Qiagen gel extraction kit. Thepurified DNA was self-ligated using T4 ligase and used to transform theE. coli strain DH5α to ampicillin resistance. Restriction enzymeanalysis of DNA recovered from several ampicillin-resistant clonesconfirmed the disruption of the DraIII site on pEG1823. The recombinantplasmid from one such clone was designated pEG2207. pEG2207 wassubsequently used to transform, via electroporation, theacrystalliferous B. thuringiensis strain EG10650 to erythromycinresistance. The recombinant strain containing pEG2207 was designatedEG12158.

Strains EG11658, EG12156, and EG12158 were used to inoculate 100 ml C2broth cultures containing 10 μg/ml erythromycin. The broth cultures weregrown with shaking in 500 ml baffled flasks at 28-30° C. for 3 days atwhich time the cultures were fully sporulated and the sporangia lysed.The spores and crystals were pelleted by centrifugation at 8,000 rpm(˜9800×g) in a JA14 rotor for 20 min at 4° C. The pellets were suspendedin 50 ml of 10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 0.005% Triton® X-100(pH 7.0). The spores and crystals were pelleted again by centrifugationat 3,750 rpm (˜3200×g) in a Beckman GPR centrifuge for 1 hour at 4° C.The pellets were resuspended in 10 ml of 10 mM Tris-HCl, 50 mM NaCl, 1mM EDTA, 0.005% Triton® X-100 (pH 7.0) and stored at 4-8° C.

Crystal proteins produced by these cultures were detected by SDS-PAGEand subsequent staining of the SDS gels with Coomassie Brilliant BlueR-250 as described in Section 5.11. The results of this analysisconfirmed that strain EG12156 produced CryET80, but not CryET76, whilestrain EG12158 produced CryET76, but not CryET80 (FIG. 3). Thus, eachcrystal protein could be produced independently of the other crystalprotein. The role of each crystal protein in effecting toxicity towardsWCRW larvae may now be studied in even greater detail.

The SDS-PAGE analysis described in FIG. 3 also revealed the presence ofan additional protein present in both the EG12156 and EG12158 crystalpreparations. This protein exhibited an apparent molecular mass ofapproximately 35 kDa and was designated CryET84.

Additional DNA sequence analysis of the cloned insert in pEG1823revealed a third open reading frame sufficient to code for a ˜38-kDaprotein (SEQ ID NO:19). This coding region is located immediately 5′ tothe cryET80 gene. Thus, cryET84, cryET80, and cryET76 may comprise anoperon. The CryET84 protein isolated from EG4851 comprises a 341-aminoacid sequence, and has a calculated molecular mass of approximately37,884 Da. CryET84 has a calculated isoelectric constant (pI) equal to5.5.

SDS-PAGE analysis of the EG11658 crystal proteins used for the WCRWbioassays described in Section 5.21 did not detect the CryET84 proteinband. Apparently, subtle differences in the cultivation of the strain orin the harvesting and washing of the spore-crystal suspension can resultin the loss of CryET84.

Sequence comparisons using Blast 2.0 and FASTA 3, as described inExample 8, revealed no significant sequence similarity between CryET84and all known B. thuringiensis crystal proteins.

5.24 Example 24 Preparation of Insect-Resistant Transgenic Plants

5.24.1 Plant Transgene Construction

The expression of a plant transgene which exists in double-stranded DNAform involves transcription of messenger RNA (mRNA) from one strand ofthe DNA by RNA polymerase enzyme, and the subsequent processing of themRNA primary transcript inside the nucleus. This processing involves a3′ non-translated region which adds polyadenylate nucleotides to the 3′end of the RNA. Transcription of DNA into mRNA is regulated by a regionof DNA usually referred to as the “promoter”. The promoter regioncontains a sequence of bases that signals RNA polymerase to associatewith the DNA and to initiate the transcription of mRNA using one of theDNA strands as a template to make a corresponding strand of RNA.

A number of promoters which are active in plant cells have beendescribed in the literature. Such promoters may be obtained from plantsor plant viruses and include, but are not limited to, the nopalinesynthase (NOS) and octopine synthase (OCS) promoters (which are carriedon tumor-inducing plasmids of Agrobacterium tumefaciens), thecauliflower mosaic virus (CaMV) 19S and 35S promoters, thelight-inducible promoter from the small subunit of ribulose1,5-bisphosphate carboxylase (ssRUBISCO, a very abundant plantpolypeptide), and the Figwort Mosaic Virus (FMV) 35S promoter. All ofthese promoters have been used to create various types of DNA constructswhich have been expressed in plants (see e.g., U.S. Pat. No. 5,463,175,specifically incorporated herein by reference).

The particular promoter selected should be capable of causing sufficientexpression of the enzyme coding sequence to result in the production ofan effective amount of protein. One set of preferred promoters areconstitutive promoters such as the CaMV35S or FMV35S promoters thatyield high levels of expression in most plant organs (U.S. Pat. No.5,378,619, specifically incorporated herein by reference). Another setof preferred promoters are root enhanced or specific promoters such asthe CaMV derived 4 as-1 promoter or the wheat POX1 promoter (U.S. Pat.No. 5,023,179, specifically incorporated herein by reference; Hertig etal., 1991). The root enhanced or specific promoters would beparticularly preferred for the control of corn rootworm (Diabroticusspp.) in transgenic corn plants.

The promoters used in the DNA constructs of the present invention may bemodified, if desired, to affect their control characteristics. Forexample, the CaMV35S promoter may be ligated to the portion of thessRUBISCO gene that represses the expression of ssRUBISCO in the absenceof light, to create a promoter which is active in leaves but not inroots. The resulting chimeric promoter may be used as described herein.For purposes of this description, the phrase “CaMV35S” promoter thusincludes variations of CaMV35S promoter, e.g., promoters derived bymeans of ligation with operator regions, random or controlledmutagenesis, etc. Furthermore, the promoters may be altered to containmultiple “enhancer sequences” to assist in elevating gene expression.

The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNA's, fromsuitable eucaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence.

For optimized expression in monocotyledenous plants such as maize, anintron may also be included in the DNA expression construct. This intronwould typically be placed near the 5′ end of the mRNA in untranslatedsequence. This intron could be obtained from, but not limited to, a setof introns consisting of the maize hsp70 intron (U.S. Pat. No.5,424,412; specifically incorporated herein by reference) or the riceAct1 intron (McElroy et al., 1990).

As noted above, the 3′ non-translated region of the chimeric plant genesof the present invention contains a polyadenylation signal whichfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. Examples of preferred 3′ regions are (1) the 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene and (2) plant genes such as the pea ssRUBISCO E9gene (Wong et al., 1992).

5.24.2 Plant Transformation and Expression

A transgene containing a δ-endotoxin coding sequence of the presentinvention can be inserted into the genome of a plant by any suitablemethod such as those detailed herein. Suitable plant transformationvectors include those derived from a Ti plasmid of A. tumefaciens, aswell as those disclosed, e.g., by Herrera-Estrella (1983), Bevan et al.(1983), Klee (1985) and Eur. Pat. Appl. Publ. No. EP0120516. In additionto plant transformation vectors derived from the Ti or root-inducing(Ri) plasmids of A. tumefaciens, alternative methods can be used toinsert the DNA constructs of this invention into plant cells. Suchmethods may involve, for example, the use of liposomes, electroporation,chemicals that increase free DNA uptake, free DNA delivery viamicroprojectile bombardment, and transformation using viruses or pollen(Fromm et al., 1986; Fromm et al., 1990). Such methods are described indetail in Section 4.0.

5.24.3 Construction of Plant Expression Vectors

For efficient expression of the polynucleotides disclosed herein intransgenic plants, the selected sequence region(s) encoding theinsecticidal polypeptide(s) must have a suitable sequence composition(Diehn et al., 1996).

For example, to place one or more of cry genes described herein in avector suitable for expression in monocotyledonous plants (e.g., undercontrol of the enhanced Cauliflower Mosaic Virus 35S promoter and linkto the hsp70 intron followed by a nopaline synthase polyadenylation siteas in U.S. Pat. No. 5,424,412, specifically incorporated herein byreference), the vector may be digested with appropriate enzymes such asNcoI and EcoRI. The larger vector band of approximately 4.6 kb is thenelectrophoresed, purified, and ligated with T4 DNA ligase to theappropriate restriction fragment containing the plantized cry gene. Theligation mix is then transformed into E. coli, carbenicillin resistantcolonies recovered and plasmid DNA recovered by DNA miniprep procedures.The DNA may then be subjected to restriction endonuclease analysis withenzymes such as NcoI and EcoRI (together), NotI, and PstI to identifyclones containing the cry gene coding sequence fused to the hsp70 intronunder control of the enhanced CaMV35S promoter).

To place the δ-endotoxin gene in a vector suitable for recovery ofstably transformed and insect resistant plants, the restriction fragmentfrom pMON33708 containing the lysine oxidase coding sequence fused tothe hsp70 intron under control of the enhanced CaMV35S promoter may beisolated by gel electrophoresis and purification. This fragment can thenbe ligated with a vector such as pMON30460 treated with NotI and calfintestinal alkaline phosphatase (pMON30460 contains the neomycinphosphotransferase coding sequence under control of the CaMV35Spromoter). Kanamycin resistant colonies may then be obtained bytransformation of this ligation mix into E. coli and colonies containingthe resulting plasmid can be identified by restriction endonucleasedigestion of plasmid miniprep DNAs. Restriction enzymes such as NotI,EcoRV, HindIII, NcoI, EcoRI, and BglII can be used to identify theappropriate clones containing the restriction fragment properly insertedin the corresponding site of pMON30460, in the orientation such thatboth genes are in tandem (i.e. the 3′ end of the cry gene expressioncassette is linked to the 5′ end of the nptII expression cassette).Expression of the Cry proteins by the resulting vector is then confirmedin plant protoplasts by electroporation of the vector into protoplastsfollowed by protein blot and ELISA analysis. This vector can beintroduced into the genomic DNA of plant embryos such as maize byparticle gun bombardment followed by paromomycin selection to obtaincorn plants expressing the cry gene essentially as described in U.S.Pat. No. 5,424,412, specifically incorporated herein by reference. Inthis example, the vector was introduced via cobombardment with ahygromycin resistance conferring plasmid into immature embryo scutella(IES) of maize, followed by hygromycin selection, and regeneration.Transgenic plant lines expressing the selected cry protein are thenidentified by ELISA analysis. Progeny seed from these events may thensubsequently tested for protection from susceptible insect feeding.

5.25 Example 25 Modification of Bacterial Genes for Expression in Plants

Many wild-type genes encoding bacterial crystal proteins are known to beexpressed poorly in plants as a full-length gene or as a truncated gene.Typically, the G+C content of a cry gene is low (37%) and often containsmany A+T rich regions, potential polyadenylation sites and numerousATTTA sequences. Table 26 shows a list of potential polyadenylationsequences which should be avoided when preparing the “plantized” geneconstruct.

TABLE 26 LIST OF SEQUENCES OF POTENTIAL POLYADENYLATION SIGNALS AATAAA*AAGCAT AATAAT* ATTAAT AACCAA ATACAT ATATAA AAAATA AATCAA ATTAAA** ATACTAAATTAA** ATAAAA AATACA** ATGAAA CATAAA** *indicates a potential majorplant polyadenylation site. **indicates a potential minor animalpolyadenylation site. All others are potential minor plantpolyadenylation sites.

The regions for mutagenesis may be selected in the following manner. Allregions of the DNA sequence of the cry gene are identified whichcontained five or more consecutive base pairs which were A or T. Thesewere ranked in terms of length and highest percentage of A+T in thesurrounding sequence over a 20-30 base pair region. The DNA is analysedfor regions which might contain polyadenylation sites or ATTTAsequences. Oligonucleotides are then designed which maximize theelimination of A+T consecutive regions which contained one or morepolyadenylation sites or ATTTA sequences. Two potential plantpolyadenylation sites have been shown to be more critical based onpublished reports. Codons are selected which increase G+C content, butdo not generate restriction sites for enzymes useful for cloning andassembly of the modified gene (e.g., BamHI, BglII, SacI, NcoI, EcoRV,etc.). Likewise condons are avoided which contain the doublets TA or GCwhich have been reported to be infrequently-found codons in plants.

Although the CaMV35S promoter is generally a high level constitutivepromoter in most plant tissues, the expression level of genes driven theCaMV35S promoter is low in floral tissue relative to the levels seen inleaf tissue. Because the economically important targets damaged by someinsects are the floral parts or derived from floral parts (e.g., cottonsquares and bolls, tobacco buds, tomato buds and fruit), it is oftenadvantageous to increase the expression of crystal proteins in thesetissues over that obtained with the CaMV35S promoter.

The 35S promoter of Figwort Mosaic Virus (FMV) is analogous to theCaMV35S promoter. This promoter has been isolated and engineered into aplant transformation vector. Relative to the CaMV promoter, the FMV 35Spromoter is highly expressed in the floral tissue, while still providingsimilar high levels of gene expression in other tissues such as leaf. Aplant transformation vector, may be constructed in which one or morefull-length native or plantized δ-endotoxin-encoding genes is driven bythe FMV 35S promoter. For example, tobacco plants may be transformedwith such a vector and compared for expression of the crystal protein(s)by Western blot or ELISA immunoassay in leaf and/or floral tissue. TheFMV promoter has been used to produce relatively high levels of crystalprotein in floral tissue compared to the CaMV promoter.

5.26 Example 26 Expression of Native or Plantized Cry Genes withssRUBISCO Promoters and Chloroplast Transit Peptides

The genes in plants encoding the small subunit of RUBISCO (SSU) areoften highly expressed, light regulated and sometimes show tissuespecificity. These expression properties are largely due to the promotersequences of these genes. It has been possible to use SSU promoters toexpress heterologous genes in transformed plants. Typically a plant willcontain multiple SSU genes, and the expression levels and tissuespecificity of different SSU genes will be different. The SSU proteinsare encoded in the nucleus and synthesized in the cytoplasm asprecursors that contain an NH₂-terminal extension known as thechloroplast transit peptide (CTP). The CTP directs the precursor to thechloroplast and promotes the uptake of the SSU protein into thechloroplast. In this process, the CTP is cleaved from the SSU protein.These CTP sequences have been used to direct heterologous proteins intochloroplasts of transformed plants.

The SSU promoters might have several advantages for expression ofheterologous genes in plants. Some SSU promoters are very highlyexpressed and could give rise to expression levels as high or higherthan those observed with the CaMV35S promoter. The tissue distributionof expression from SSU promoters is different from that of the CaMV35Spromoter, so for control of some insect pests, it may be advantageous todirect the expression of crystal proteins to those cells in which SSU ismost highly expressed. For example, although relatively constitutive, inthe leaf the CaMV35S promoter is more highly expressed in vasculartissue than in some other parts of the leaf, while most SSU promotersare most highly expressed in the mesophyll cells of the leaf. Some SSUpromoters also are more highly tissue specific, so it could be possibleto utilize a specific SSU promoter to express the protein of the presentinvention in only a subset of plant tissues, if for example expressionof such a protein in certain cells was found to be deleterious to thosecells. For example, for control of Colorado potato beetle in potato, itmay be advantageous to use SSU promoters to direct crystal proteinexpression to the leaves but not to the edible tubers.

Utilizing SSU CTP sequences to localize crystal proteins to thechloroplast might also be advantageous. Localization of the B.thuringiensis crystal proteins to the chloroplast could protect thesefrom proteases found in the cytoplasm. This could stabilize the proteinsand lead to higher levels of accumulation of active toxin. cry genescontaining the CTP may be used in combination with the SSU promoter orwith other promoters such as CaMV35S.

5.27 Example 27 Targeting of δ-Endotoxin Polypeptides to theExtracellular Space or Vacuole Using Signal Peptides

The B. thuringiensis δ-endotoxin polypeptides described herein mayprimarily be localized to the cytoplasm of transformed plant cell, andthis cytoplasmic localization may result in plants that areinsecticidally-resistant. However, in certain embodiments, it may beadvantageous to direct the localization or production of the B.thuringiensis polypeptide(s) to one or more compartments of a plant, orto particular types of plant cells. Localizing B. thuringiensis proteinsin compartments other than the cytoplasm may result in less exposure ofthe B. thuringiensis proteins to cytoplasmic proteases leading togreater accumulation of the protein yielding enhanced insecticidalactivity. Extracellular localization could lead to more efficientexposure of certain insects to the B. thuringiensis proteins leading togreater efficacy. If a B. thuringiensis protein were found to bedeleterious to plant cell function, then localization to anoncytoplasmic compartment could protect these cells from the toxicityof the protein.

In plants as well as other eukaryotes, proteins that are destined to belocalized either extracellularly or in several specific compartments aretypically synthesized with an NH₂-terminal amino acid extension known asthe signal peptide. This signal peptide directs the protein to enter thecompartmentalization pathway, and it is typically cleaved from themature protein as an early step in compartmentalization. For anextracellular protein, the secretory pathway typically involvescotranslational insertion into the endoplasmic reticulum with cleavageof the signal peptide occurring at this stage. The mature protein thenpasses through the Golgi body into vesicles that fuse with the plasmamembrane thus releasing the protein into the extracellular space.Proteins destined for other compartments follow a similar pathway. Forexample, proteins that are destined for the endoplasmic reticulum or theGolgi body follow this scheme, but they are specifically retained in theappropriate compartment. In plants, some proteins are also targeted tothe vacuole, another membrane bound compartment in the cytoplasm of manyplant cells. Vacuole targeted proteins diverge from the above pathway atthe Golgi body where they enter vesicles that fuse with the vacuole.

A common feature of this protein targeting is the signal peptide thatinitiates the compartmentalization process. Fusing a signal peptide to aprotein will in many cases lead to the targeting of that protein to theendoplasmic reticulum. The efficiency of this step may depend on thesequence of the mature protein itself as well. The signals that direct aprotein to a specific compartment rather than to the extracellular spaceare not as clearly defined. It appears that many of the signals thatdirect the protein to specific compartments are contained within theamino acid sequence of the mature protein. This has been shown for somevacuole targeted proteins, but it is not yet possible to define thesesequences precisely. It appears that secretion into the extracellularspace is the “default” pathway for a protein that contains a signalsequence but no other compartmentalization signals. Thus, a strategy todirect B. thuringiensis proteins out of the cytoplasm is to fuse thegenes for synthetic B. thuringiensis genes to DNA sequences encodingknown plant signal peptides. These fusion genes will give rise to B.thuringiensis proteins that enter the secretory pathway, and lead toextracellular secretion or targeting to the vacuole or othercompartments.

Signal sequences for several plant genes have been described. One suchsequence is for the tobacco pathogenesis related protein PR1b has beenpreviously described (Cornelissen et al., 1986). The PR1b protein isnormally localized to the extracellular space. Another type of signalpeptide is contained on seed storage proteins of legumes. These proteinsare localized to the protein body of seeds, which is a vacuole likecompartment found in seeds. A signal peptide DNA sequence for theβ-subunit of the 7S storage protein of common bean (Phaseolus vulgaris),PvuB has been described (Doyle et al., 1986). Based on the publishedthese published sequences, genes may be synthesized chemically usingoligonucleotides that encode the signal peptides for PR1b and PvuB. Insome cases to achieve secretion or compartmentalization of heterologousproteins, it may be necessary to include some amino acid sequence beyondthe normal cleavage site of the signal peptide. This may be necessary toinsure proper cleavage of the signal peptide.

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims. Accordingly, the exclusive rights sought to be patentedare as described in the claims below.

1. A Bacillus thuringiensis cell having the NRRL accession number NRRLB-21915 or NRRL B-21916.
 2. An isolated polynucleotide encoding SEQ IDNO:2.
 3. The polynucleotide of claim 2, wherein said polynucleotidefurther encodes SEQ ID NO:4.
 4. The polynucleotide of claim 3,comprising SEQ ID NO:1 and SEQ ID NO:3.
 5. The polynucleotide of claim3, wherein said polynucleotide further encodes SEQ ID NO:19.
 6. Thepolynucleotide of claim 5, comprising SEQ ID NO:1, SEQ ID NO:3 and SEQID NO:18.
 7. The polynucleotide of claim 2, comprising SEQ ID NO:1. 8.The polynucleotide of claim 2, further characterized as RNA or DNA. 9.The polynucleotide of claim 2, wherein said isolated polynucleotide isoperably linked to a promoter.
 10. The polynucleotide of claim 9,wherein said promoter is a heterologous promoter.
 11. A nucleic acidvector comprising the polynucleotide of claim
 2. 12. The vector of claim11, wherein said polynucleotide further encodes SEQ ID NO:4.
 13. Thevector of claim 12, wherein said polynucleotide further encodes SEQ IDNO:19.
 14. The vector of claim 11, further defined as a plasmid,baculovirus, artificial chromosome, virion, cosmid, phagemid, phage orviral vector.
 15. A transformed host cell comprising the polynucleotideof claim
 2. 16. The transformed host cell of claim 15, wherein thepolynucleotide further encodes SEQ ID NO:4.
 17. The transformed hostcell of claim 16, wherein said polynucleotide further encodes SEQ IDNO:19.
 18. The transformed host cell of claim 15, further defined as aprokaryotic host cell.
 19. The transformed host cell of claim 18,further defined as a bacterial cell.
 20. The transformed host cell ofclaim 19, wherein said bacterial cell is a Bacillus thuringiensis,Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Escherichia,Salmonella, Agrobacterium or Pseudomonas cell.
 21. The transformed hostcell of claim 20, wherein said bacterial cell is a Bacillusthuringiensis NRRL B-21915 or NRRL B-21916 cells.
 22. The transformedhost cell of claim 20, wherein said bacterial cell is an Agrobacteriumtumefaciens cell.